Amine-containing lipidoids and uses thereof

ABSTRACT

Provided herein are lipidoids that may be prepared from the conjugate addition of alkylamines to acrylates. In some embodiments, provided lipidoids are biodegradable and may be used in a variety of drug delivery systems. Given the amino moiety of the lipidoids, they are well-suited for the delivery of polynucleotides, in addition to other agents. Nanoparticles containing the inventive lipidoids and polynucleotides have been prepared and have been shown to be effective in delivering siRNA.

RELATED REFERENCES

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. patent application, U.S. Ser. No. 13/966,136,filed Aug. 13, 2013, which claims priority under 35 U.S.C. §119(e) toU.S. provisional application, U.S. Ser. No. 61/682,468, filed Aug. 13,2012, which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under GrantNo.5-F32-EB009623-02 awarded by the National Institutes of Health. TheU.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The discovery of RNA interference (RNAi) in mammalian cells (Fire, etal. Nature 391:806-811 (1998)) has allowed for the development of shortinterfering RNA (siRNA) therapeutics (Elbashir, et al. Nature 411:494-8(2001)), which have the potential to treat a wide variety of humandiseases, including viral infections and cancer, through geneticmodulation. Theoretically, siRNA can be used to alter the expression ofnearly any gene in the body through the silencing of complementarymessenger RNA. Such precise genetic control offers a broad therapeuticpotential that is typically not attainable using conventional smallmolecule drugs. siRNA delivery vehicles must negotiate a number ofobstacles in vivo prior to delivering their payload to target cells. Inaddition to escorting therapeutic cargo through the bloodstream andextracellular matrix, delivery vehicles must mediate siRNA transportacross the cellular membrane of the target cell as well as to facilitateendosomal escape prior to lysosomal digestion (Akinc, et al. J. Gene.Med. 7:657-63 (2005)). It is only once these barriers have been breachedthat siRNA can interact with the RNAi machinery within the cytoplasm andtrigger the gene silencing process (Whitehead, et al. Nature Rev. DrugDiscov. 8:129-38 (2009)).

A select number of delivery systems have previously been reported todeliver siRNA for the treatment of a variety of disease targets in vivo,including hypercholesterolemia (Frank-Kamenetsky, et al. Proc. Natl.Acad. Sci. USA 107:1864-9 (2010); Love, et al. Proc. Natl. Acad. Sci.USA 26:431-42 (2008)), liver cirrhosis (Sato, et al. Nature Biotechnol.26:431-42 (2008)), Ebola virus (Geisbert, et al. Lancet 375:1896-1905(2010)), and cancer (Huang, et al. Proc. Natl. Acad. Sci. USA106:3426-30 (2009)). Unfortunately, RNAi success in vivo has notconsistently translated to success in the clinic. Because siRNA must bedosed repeatedly to achieve therapeutic effect, ideal delivery vehicleswill offer a substantial therapeutic window in order to ensure thebroadest clinical application. Although some materials have beenidentified that allow for potent gene silencing at siRNA doses as low as0.01 mg/kg (Love, et al. Proc. Natl. Acad. Sci. USA 107:1864-9 (2010)),their clinical potential has been limited due to a lack of deliveryvehicle degradability. There exists a continuing need for non-toxic,biodegradable, biocompatible lipids that can be used to transfectnucleic acids and other therapeutic agents. Such lipids would haveseveral uses, including the delivery of siRNA.

SUMMARY OF THE INVENTION

The compounds described herein, known as lipidoids for their lipid-liketails, may be prepared by the addition of a primary or secondary amineto an acrylate via a Michael addition reaction. The lipidoids describedherein may be used in the delivery of therapeutic agents to a subject.The inventive lipidoids are particularly useful in delivering negativelycharged agents. For example, lipidoids described herein may be used todeliver DNA, RNA, or other polynucleotides to a subject or to a cell. Incertain embodiments, lipidoids of the present invention are used todeliver siRNA. In certain embodiments, lipidoids described herein areuseful as reagents.

In one aspect, the present invention provides a compound of the Formula(I):

or a salt thereof, wherein L, R, R^(A), and q are as defined herein. Incertain embodiments, a provided compound is of the Formula (I-a), (I-b),(I-c), (I-d), or (I-e):

or a salt thereof, wherein m, n, R, and R^(A) are as defined herein.

In another aspect, the present invention provides a compound of theFormula (II):

or a salt thereof, wherein L, R^(C), and R^(A) are as defined herein. Incertain embodiments, a provided compound is of the Formula (II-a),(II-b), (II-c), (II-d), (II-e), or (II-f):

or a salt thereof, wherein v, L, R, R^(D), and R^(A) are as definedherein.

In another aspect, the present invention provides a compound of theFormula (III):

or a salt thereof, wherein p, R¹, j, and R are as defined herein. Incertain embodiments, a provided compound is of the Formula (III-a),(III-b), (III-c), (III-d), or (III-e):

or a salt thereof, wherein w, p, R¹, j, and R are as defined herein.

In another aspect, the present invention provides a compound of Formula(IV)

or a salt thereof, wherein R, x, and y are as defined herein. In certainembodiments, a provided compound is of the Formula (IV-a):

or a salt thereof, wherein R is as defined herein.

In another aspect, the present invention provides a compound of Formula(V):

or a salt thereof, wherein L, R², g, and R are as defined herein. Incertain embodiments, a provided compound is of the Formula (V-a), (V-b),(V-c), or (V-d):

or a salt thereof, wherein L, R², g, and R are as defined herein.

In another aspect, the present invention provides a compound of formula

or a salt thereof, wherein R and R^(A) are as defined herein.

In another aspect, the present invention provides lipidoids havingcertain features. In some embodiments, a lipidoid of the presentinvention is prepared from an alkylamine starting material that has atleast one tertiary amine. In some embodiments, a lipidoid of the presentinvention has three or more lipid-like tails. In some embodiments, thelipid-like tails on a lipidoid of the present invention are betweenC₁₂-C₁₄ in length, e.g., C₁₃ (e.g., derived from the O₁₃ acrylate shownin FIG. 1). In certain embodiments, a provided lipidoid is prepared froman alkylamine starting material that has at least one tertiary amine andhas three or more C₁₃ tails.

In another aspect, the inventive lipidoids are combined with an agent toform nanoparticles, microparticles, liposomes, or micelles. The agent tobe delivered by the nanoparticles, microparticles, liposomes, ormicelles may be in the form of a gas, liquid, or solid, and the agentmay be, for example, a polynucleotide, protein, peptide, or smallmolecule. In certain embodiments, inventive lipidoids may be combinedwith other lipids, polymers, surfactants, cholesterol, carbohydrates,proteins, etc. to form the particles. In certain embodiments, theparticles may be combined with an excipient to form pharmaceutical orcosmetic compositions.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools or probes in biological assays.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons. In someembodiments, an aliphatic group is optionally substituted with one ormore functional groups. As will be appreciated by one of ordinary skillin the art, “aliphatic” is intended herein to include alkyl, alkenyl,alkynyl, cycloalkyl, and cycloalkenyl moieties.

The term “alkyl” as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. Examples of alkyl radicals include, but are not limitedto, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl,neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

In certain embodiments, the alkyl groups employed in the inventivelipidoids contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl groups employed in the inventive lipidoidscontain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkylgroups contain 1-8 aliphatic carbon atoms. In still other embodiments,the alkyl groups employed in the invention contain 1-6 aliphatic carbonatoms. In yet other embodiments, the alkyl groups contain 1-4 carbonatoms. Illustrative alkyl groups thus include, but are not limited to,for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl,n-hexyl, and sec-hexyl.

The terms “alkenyl” and “alkynyl” are given their ordinary meaning inthe art and refer to unsaturated aliphatic groups analogous in lengthand possible substitution to the alkyls described above, but thatcontain at least one double or triple bond respectively.

The term “cycloalkyl”, as used herein, refers saturated, cyclichydrocarbon radicals derived from a hydrocarbon moiety containingbetween three and seven carbon atoms by removal of a single hydrogenatom. Suitable cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cycloheptyl.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupattached via a straight chain or branched alkyl group. Suitablecycloalkylalkyl groups include, but are not limited to,—CH₂(cyclopropyl), —CH₂CH₂(cyclopropyl), —CH₂(cyclobutyl),—CH₂CH₂(cyclobutyl), —CH₂(cyclopentyl), —CH₂CH₂(cyclopentyl),—CH₂(cyclohexyl), —CH₂CH₂(cyclohexyl), —CH₂(cycloheptyl), and—CH₂CH₂(cycloheptyl).

The term “alkylene” as used herein refers to a bivalent alkyl group. An“alkylene” group is a polymethylene group, i.e., —(CH₂)_(k)—, wherein kis a positive integer, e.g., from 1 to 20, from 1 to 10, from 1 to 6,from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. In someembodiments, one or more hydrogens on an alkylene group is replaced by asubstituent (e.g., fluoro).

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, including, for example, mammals, birds, reptiles,amphibians, and fish. Preferably, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, aprimate, or a pig). An animal may be a transgenic animal.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. Preferably, the association is covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe compounds that are not toxic to cells. In certain embodiments,compounds are “biocompatible” if their addition to cells in vitro at aminimum therapeutically effective dose results in less than or equal to20% cell death, and their administration in vivo does not induceinflammation or other such adverse effects.

“Biodegradable”: As used herein, “biodegradable” compounds are thosethat, when introduced into cells, are broken down by the cellularmachinery or by hydrolysis into components that the cells can eitherreuse or dispose of without significant long-term toxic effect on thecells. In certain embodiments, the components do not induce inflammationor other adverse effects in vivo. In certain embodiments, the chemicalreactions relied upon to break down the biodegradable compounds areuncatalyzed.

“Peptide” or “protein”: According to the present invention, a “peptide”or “protein” comprises a string of at least three amino acids linkedtogether by peptide bonds. The terms “protein” and “peptide” may be usedinterchangeably. Peptide may refer to an individual peptide or acollection of peptides. Inventive peptides preferably contain onlynatural amino acids, although non-natural amino acids (i.e., compoundsthat do not occur in nature but that can be incorporated into apolypeptide chain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in aninventive peptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. In apreferred embodiment, the modifications of the peptide lead to a morestable peptide (e.g., greater half-life in vivo). These modificationsmay include cyclization of the peptide, the incorporation of D-aminoacids, etc. None of the modifications should substantially interferewith the desired biological activity of the peptide.

“Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotiderefers to a polymer of nucleotides. Typically, a polynucleotidecomprises at least three nucleotides. The polymer may include naturalnucleosides (i.e., adenosine, thymidine, guano sine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

“Small molecule”: As used herein, the term “small molecule” refers toorganic compounds, whether naturally-occurring or artificially created(e.g., via chemical synthesis) that have relatively low molecular weightand that are not proteins, polypeptides, or nucleic acids. Typically,small molecules have a molecular weight of less than about 1500 g/mol.Also, small molecules typically have multiple carbon-carbon bonds. Knownnaturally-occurring small molecules include, but are not limited to,penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Knownsynthetic small molecules include, but are not limited to, ampicillin,methicillin, sulfamethoxazole, sulfonamides, dexamethasone, anddoxorubicin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays a subset of the large library of biodegradable lipidoidsthat were synthesized combinatorially through the conjugate addition ofalkylamines (in red) to alkyl-acrylate tails (in blue). The rest of thealkylamines used in lipidoid library synthesis are shown in FIG. 2.

FIG. 2 shows additional alkylamines used in the lipidoid library.

FIG. 3 shows the evaluation of lipidoids for an ability to deliver siRNAto HeLa cells. (a) Relative luciferase activity values (firefly lucifaseactivity normalized to control Renilla luciferase activity) are shownfor 1400 lipidoids. ˜7% of the library induced >50% gene silencing(shown in red). The tail length (b), tail substitution number (c) andalkyl-amine composition (d) influenced in vitro activity.

FIG. 4 demonstrates that select lipidoids induced a high degreesilencing of multiple targets in mice. (a) Of the ˜100 lipidoids testedin vivo, 15 induced complete Factor VII knockdown in mouse hepatocytesat a total siRNA dose of 5 mg/kg (data points in red). (b) The EC₅₀values of these top 15 lipidoids ranged from 0.05 to 1.5 mg/kg understandard formulation conditions. (c) The amount of PEG in the lipidnanoparticle formulation had a dramatic effect on efficacy. Data isshown for the lipidoid 304O₁₄, which produced the most efficaciousformulation of the study when formulated with 0.75% PEG. (d) Doseresponse and Factor VII activity recovery data for the optimized 304O₁₃lipid nanoparticle formulation. 304O₁₃ also induced CD45 silencing inmonocyte and macrophage (CD11b+) populations in the peritoneal cavity(e) as well as in dendritic cells (CD11c+) in the spleen 3 dayspost-injection. In all panels, error bars represent standard deviation(n=3).

FIG. 5 shows biodistribution images for Cy5.5-labeled siRNA deliveredwith the lipidoid 304O₁₃. IVIS (a) and Odyssey (b) imaging show that,while naked siRNA is primarily cleared through the kidneys, 304O₁₃mediates accumulation in the liver and spleen. (c) Confocal microscopyof 304O₁₃-treated liver shows siRNA (red) delivery into nearly allcells, including Kupffer cells and hepatocytes. In contrast, naked siRNAhad a limited penetration depth from the blood vessels intohepatocellular tissue. (d) 304O₁₃ lipid nanoparticles were rapidlyeliminated from the bloodstream after tail vein injection. Error barsrepresent standard deviation (n=3).

FIG. 6 shows a comparison of (a) cytokine profiles 4 hourspost-injection and (b) liver histology sections 72 hours post-injectionfor degradable (304O₁₃) and non-degradable (C12-200) lipidnanoparticles.

FIG. 7 displays structure-function information of efficacious lipidnanoparticles. (a) Of the 108 lipid nanoparticles tested for siRNAdelivery to hepatocytes in mice, 66 had 3 or more tails, 42 had atertiary amine present in the original alkyl-amine, and 25 had an O₁₃tail length. 88% of the lipid nanoparticles exhibiting all three“efficacy criteria” achieved complete FVII knockdown. The percentage ofefficacious lipid nanoparticles decreased precipitously when anycriterion were not met. (b) Twelve second generation lipid nanoparticleswere made to meet all efficacy criteria by first synthesizing customalkyl-amines and reacting them with O₁₃ tails. (c) 83% of secondgeneration LNPs achieved complete FVII silencing in vivo, and (d) EC₅₀sunder non-optimized LNP formulating conditions ranged from 0.05 to 1mg/kg total siRNA. (e) 503O₁₃ was the most efficacious LNP uponformulation, with an EC₅₀ of 0.01 mg/kg. 503O₁₃ encapsulating controlsiRNA did not result in FVII knockdown. Error bars represent standarddeviation (n=3).

FIG. 8 shows that lipid nanoparticles that induced complete FVIIsilencing at 5 mg/kg behaved in a dose-dependent manner. Each lipidnanoparticle was evaluated at three additional doses (2, 0.5, and 0.1mg/kg) shown from left to right. Error bars represent standard deviation(n=3).

FIG. 9 shows that the lipid nanoparticles 306O₁₂, 306O₁₄ and 315O₁₂facilitated modest silencing of the surface receptor CD45 in variouswhite blood cell populations harvested from the peritoneal cavity (left)and spleen (right) of B6 mice three days post-injection (dose=2.5 mg/kgtotal siRNA). Error bars represent standard deviation (n=3).

FIG. 10 shows that pKa values significantly influence delivery efficacyto hepatocytes in vivo. All lipidoid nanoparticles capable of mediatingcomplete Factor VII gene silencing had pKa values greater or equal to5.5.

FIGS. 11A and 11B show degradation by hydrolysis of the lipidoid 304O₁₃.Overlay of ¹H NMR spectra of the starting material 304O₁₃, the crudereaction mixture, and authentic 1-tridecanol demonstrated that the304O₁₃ had been consumed and that tridecanol had been formed insignificant quantity under both acidic and basic conditions. FIG. 11Ashows acidic hydrolysis condition, and FIG. 11B shows basic hydrolysiscondition.

FIG. 12 shows that clinical chemistry parameters were evaluated fornegative control (PBS, black), 304O₁₃ (blue), and C12-200 (red) groupsof C57BL/6 mice. The mice had been injected with either a single 3 mg/kgdose of total siRNA or four 3 mg/kg doses (lx per week for four weeks).Blood was drawn for analysis 72 hours post-final injection. There wereno statistically significant changes in any of the clinical chemistryparameters for any of the treated groups compared to controls (asevaluated by a student t-test). Error bars represent standard deviation(n=3-5).

FIG. 13 shows that the second generation lipid nanoparticles (LNPs)facilitated silencing of the surface receptor CD45 in various whiteblood cell populations harvested from the peritoneal cavity (left) andspleen (right) of B6 mice three days post-injection (dose=2.5 mg/kgtotal siRNA). Percent silencing was calculated by comparing to anidentically defined cell population from animals injected with anon-targeting siRNA formulated with the same LNP. Error bars representstandard deviation (n=3).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides lipidoids and lipidoid-based deliverysystems. The systems described herein may be used in thepharmaceutical/drug delivery arts to delivery polynucleotides, proteins,small molecules, peptides, antigen, drugs, etc. to a patient, tissue,organ, cell, etc.

The lipidoids of the present invention provide for several differentuses in the drug delivery art. The lipidoids with their amine-containinghydrophilic portion may be used to complex polynucleotides and therebyenhance the delivery of polynucleotides and prevent their degradation.The lipidoids may also be used in the formation of nanoparticles,microparticles, liposomes, and micelles containing the agent to bedelivered. In certain embodiments, the lipids are biocompatible andbiodegradable, and particles formed therefrom are also biodegradable andbiocompatible and may be used to provide controlled, sustained releaseof the agent. Provided lipidoids and their corresponding particles mayalso be responsive to pH changes given that these lipids are protonatedat lower pH.

Lipidoids

The lipidoids of the present invention contain primary, secondary, ortertiary amines and salts thereof. In certain embodiments, the inventivelipidoids are biodegradable. In certain embodiments, inventive lipidoidsare effective at delivering an agent (e.g., RNA) to a cell.

In certain embodiments, a lipidoid of the present invention is preparedfrom an alkylamine starting material that has at least one tertiaryamine. In some embodiments, a lipidoid of the present invention hasthree or more lipid-like tails. In some embodiments, the lipid-liketails on a lipidoid of the present invention are between C₁₀-C₁₄ inlength, e.g., C₁₂-C₁₄, e.g., C₁₃. In certain embodiments, a providedlipidoid is prepared from an alkylamine starting material that has atleast one tertiary amine and the lipidoid formed therefrom has three ormore C₁₃ tails. In certain embodiments, a provided lipidoid is preparedfrom an alkylamine starting material that has at least one tertiaryamine, provided that the amine is not amine 110, amine 113, or amine115, and the lipidoid formed therefrom has three or more C₁₃ tails.

In certain embodiments, a lipidoid of the present invention is of theFormula (I):

or a salt thereof, wherein

-   -   each L is, independently, branched or unbranched C₁₋₆ alkylene,        wherein L is optionally substituted with one or more fluorine        radicals;

each R^(A) is, independently, branched or unbranched C₁₋₆ alkyl, C₃₋₇cycloalkyl, or branched or unbranched C₄₋₁₂ cycloalkylalkyl, whereinR^(A) is optionally substituted with one or more fluorine radicals;

each R is, independently, hydrogen or —CH₂CH₂C(═O)OR^(B);

each R^(B) is, independently, C₁₀₋₁₄ alkyl, wherein R^(B) is optionallysubstituted with one or more fluorine radicals; and

q is 1, 2, or 3.

In certain embodiments, a lipidoid of formula (I) is not

As defined generally above, each L is, independently, branched orunbranched C₁₋₆ alkylene, wherein L is optionally substituted with oneor more fluorine radicals. In some embodiments, L is substituted withone or more fluorine radicals. In other embodiments, L is unsubstituted.In some embodiments, L is branched. In other embodiments, L isunbranched. In certain embodiments, L is C₁₋₄ alkylene. In certainembodiments, L is methylene, ethylene, or propylene.

As defined generally above, each R^(A) is, independently, branched orunbranched C₁₋₆ alkyl, C₃₋₇ cycloalkyl, or branched or unbranched C₄₋₁₂cycloalkylalkyl, wherein R^(A) is optionally substituted with one ormore fluorine radicals. In some embodiments, R^(A) is substituted withone or more fluorine radicals. For example, when R^(A) is methyl, it maybe substituted with one, two, or three fluorine radicals to give —CH₂F,—CHF₂, or —CF₃. In other embodiments, R^(A) is unsubstituted. In someembodiments, all R^(A) groups are the same. In other embodiments, theR^(A) groups are different. In some embodiments, R^(A) is branched orunbranched C₁₋₆ alkyl. In certain embodiments, R^(A) is branched C₁₋₆alkyl. In certain embodiments, R^(A) is unbranched C₁₋₆ alkyl. Incertain embodiments, R^(A) is C₁₋₃ alkyl. In certain embodiments, R^(A)is methyl, ethyl, or propyl. In certain embodiments, R^(A) is C₃₋₇cycloalkyl. In certain embodiments, R^(A) is cyclohexyl. In certainembodiments, R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl. Incertain embodiments, R^(A) is cycloheptyl. In some embodiments, R^(A) isbranched or unbranched C₄₋₁₂ cycloalkylalkyl.

As defined generally above, each R is, independently, hydrogen or—CH₂CH₂C(═O)OR^(B). In some embodiments, at least three R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least four R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, all R groups are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each R^(B) is, independently, C₁₀₋₁₄ alkyl,wherein R^(B) is optionally substituted with one or more fluorineradicals. In some embodiments, R^(B) is substituted with one or morefluorine radicals. For example, in some embodiments, R^(B) may besubstituted with one fluoro, or in other embodiments, may beperfluorinated. In other embodiments, R^(B) is unsubstituted. In someembodiments, all R^(B) groups are the same. In certain embodiments,R^(B) is C₁₀ alkyl. In some embodiments, R^(B) is n-decyl. In certainembodiments, R^(B) is C₁₁ alkyl. In some embodiments, R^(B) isn-undecyl. In certain embodiments, R^(B) is C₁₂ alkyl. In someembodiments, R^(B) is n-dodecyl. In certain embodiments, R^(B) is C₁₃alkyl. In some embodiments, R^(B) is n-tridecyl. In certain embodiments,R^(B) is C₁₄ alkyl. In some embodiments, R^(B) is n-tetradecyl.

As defined generally above, q is 1, 2, or 3. In some embodiments, qis 1. In some embodiments, q is 2. In some embodiments, q is 3.

In some embodiments, a lipidoid of the present invention is of theFormula (I-a):

or a salt thereof,wherein R and R^(A) are as defined above and described herein;

each n is, independently, 0, 1, or 2; and

m is 0, 1, or 2.

In some embodiments, m is 0. In some embodiments, m is 1. In someembodiments, m is 2.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, n is 2.

In some embodiments, m is 0, and n is 0. In some embodiments, m is 1,and n is 0. In some embodiments, m is 2, and n is 0. In someembodiments, m is 0, and n is 1. In some embodiments, m is 0, and n is2. In some embodiments, m is 1, and n is 1.

In some embodiments, a lipidoid of the present invention is of theFormula (I-b):

or a salt thereof, wherein R and R^(A) are as defined above anddescribed herein.

In some embodiments, a lipidoid of the present invention is of theFormula (I-c):

or a salt thereof, wherein R and R^(A) are as defined above anddescribed herein.

In some embodiments, a lipidoid of the present invention is of theFormula (I-d):

or a salt thereof, wherein R and R^(A) are as defined above anddescribed herein.

In some embodiments, a lipidoid of the present invention is of theFormula (I-e):

or a salt thereof, wherein R and R^(A) are as defined above anddescribed herein.

In some embodiments, a lipidoid of the present invention is of one ofthe following formulae:

In certain embodiments, a lipidoid of the present invention is of theFormula (II):

or a salt thereof,

-   wherein

each L is, independently, branched or unbranched C₁₋₆ alkylene, whereinL is optionally substituted with one or more fluorine radicals;

each R^(A) is, independently, branched or unbranched C₁₋₆ alkyl, C₃₋₇cycloalkyl, or branched or unbranched C₄₋₁₂ cycloalkylalkyl, whereinR^(A) is optionally substituted with one or more fluorine radicals;

each R^(C) is, independently, -L-N(R^(D))₂ or —R;

each R is, independently, hydrogen or —CH₂CH₂C(═O)OR^(B);

each R^(D) is, independently, —R^(A) or —R; and

each R^(B) is, independently, C₁₀₋₁₄ alkyl, wherein R^(B) is optionallysubstituted with one or more fluorine radicals.

As defined generally above, each L is, independently, branched orunbranched C₁₋₆ alkylene, wherein L is optionally substituted with oneor more fluorine radicals. In some embodiments, L is substituted withone or more fluorine radicals. In other embodiments, L is unsubstituted.In some embodiments, L is branched. In other embodiments, L isunbranched. In certain embodiments, L is C₁₋₄ alkylene. In certainembodiments, L is methylene, ethylene, or propylene.

As defined generally above, each R^(A) is, independently, branched orunbranched C₁₋₆ alkyl, C₃₋₇ cycloalkyl, or branched or unbranched C₄₋₁₂cycloalkylalkyl, wherein R^(A) is optionally substituted with one ormore fluorine radicals. In some embodiments, R^(A) is substituted withone or more fluorine radicals. For example, when R^(A) is methyl, it maybe substituted with one, two, or three fluorine radicals to give —CH₂F,—CHF₂, or —CF₃. In other embodiments, R^(A) is unsubstituted. In someembodiments, all R^(A) groups are the same. In other embodiments, theR^(A) groups are different. In some embodiments, R^(A) is branched orunbranched C₁₋₆ alkyl. In certain embodiments, R^(A) is branched C₁₋₆alkyl. In certain embodiments, R^(A) is unbranched C₁₋₆ alkyl. Incertain embodiments, R^(A) is C₁₋₃ alkyl. In certain embodiments, R^(A)is methyl, ethyl, or propyl. In certain embodiments, R^(A) is C₃₋₇cycloalkyl. In certain embodiments, R^(A) is cyclohexyl. In certainembodiments, R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl. Incertain embodiments, R^(A) is cycloheptyl. In some embodiments, R^(A) isbranched or unbranched C₄₋₁₂ cycloalkylalkyl.

As defined generally above, each R^(C) is, independently, -L-N(R^(D))₂or —R. In some embodiments, all R^(C) groups are —R. In someembodiments, R^(C) is -L-N(R^(D))₂.

As defined generally above, each R^(D) is, independently, —R^(A) or —R.In some embodiments, all R^(D) groups are —R. In some embodiments, oneR^(D) on a nitrogen is —R, and the other is —R^(A).

As defined generally above, each R is, independently, hydrogen or—CH₂CH₂C(═O)OR^(B). In some embodiments, at least one R group is—CH₂CH₂C(═O)OR^(B). In some embodiments, at least two R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least three R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least four R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, all R groups are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each R^(B) is, independently, C₁₀₋₁₄ alkyl,wherein R^(B) is optionally substituted with one or more fluorineradicals. In some embodiments, R^(B) is substituted with one or morefluorine radicals. For example, in some embodiments, R^(B) may besubstituted with one fluoro, or in other embodiments, may beperfluorinated. In other embodiments, R^(B) is unsubstituted. In someembodiments, all R^(B) groups are the same. In certain embodiments,R^(B) is C₁₀ alkyl. In some embodiments, R^(B) is n-decyl. In certainembodiments, R^(B) is C₁₁ alkyl. In some embodiments, R^(B) isn-undecyl. In certain embodiments, R^(B) is C₁₂ alkyl. In someembodiments, R^(B) is n-dodecyl. In certain embodiments, R^(B) is C₁₃alkyl. In some embodiments, R^(B) is n-tridecyl. In certain embodiments,R^(B) is C₁₄ alkyl. In some embodiments, R^(B) is n-tetradecyl.

In certain embodiments, a lipidoid of the present invention is of theFormula (II-a):

or a salt thereof,

-   wherein

each v is, independently, 1, 2, or 3.

In certain embodiments, v is 1. In certain embodiments, v is 2. Incertain embodiments, v is 3.

In certain embodiments, a lipidoid of the present invention is of theFormula (II-b):

or a salt thereof, wherein R^(A) and R^(B) are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is of theformula:

or a salt thereof, wherein R^(B) is as defined above and describedherein.

In certain embodiments, a lipidoid of the present invention is of theFormula (II-c):

or a salt thereof, wherein L, R^(A), and R^(D) are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (II-d):

or a salt thereof, wherein L, R^(A), and R are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (II-e):

or a salt thereof, wherein L, R^(A), and R are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (II-f):

or a salt thereof, wherein R^(A) and R are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is of theformula:

or a salt thereof, wherein R is as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (III):

or a salt thereof,

-   wherein

each L is, independently, branched or unbranched C₁₋₆ alkylene, whereinL is optionally substituted with one or more fluorine radicals;

each R is, independently, hydrogen or —CH₂CH₂C(═O)OR^(B);

each R^(B) is, independently, C₁₀₋₁₄ alkyl, wherein R^(B) is optionallysubstituted with one or more fluorine radicals;

each R¹ is, independently, fluoro or C₁₋₆ alkyl optionally substitutedwith one or more fluorine radicals;

j is 0, 1, 2, 3, or 4; and

p is 1 or 2.

In certain embodiments, at least three R groups of formula (III) are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each L is, independently, branched orunbranched C₁₋₆ alkylene, wherein L is optionally substituted with oneor more fluorine radicals. In some embodiments, L is substituted withone or more fluorine radicals. In other embodiments, L is unsubstituted.In some embodiments, L is branched. In other embodiments, L isunbranched. In certain embodiments, L is C₁₋₄ alkylene. In certainembodiments, L is methylene, ethylene, or propylene.

As defined generally above, each R is, independently, hydrogen or—CH₂CH₂C(═O)OR^(B). In some embodiments, at least three R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least four R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, all R groups are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each R^(B) is, independently, C₁₀₋₁₄ alkyl,wherein R^(B) is optionally substituted with one or more fluorineradicals. In some embodiments, R^(B) is substituted with one or morefluorine radicals. For example, in some embodiments, R^(B) may besubstituted with one fluoro, or in other embodiments, may beperfluorinated. In other embodiments, R^(B) is unsubstituted. In someembodiments, all R^(B) groups are the same. In certain embodiments,R^(B) is C₁₀ alkyl. In some embodiments, R^(B) is n-decyl. In certainembodiments, R^(B) is C₁₁ alkyl. In some embodiments, R^(B) isn-undecyl. In certain embodiments, R^(B) is C₁₂ alkyl. In someembodiments, R^(B) is n-dodecyl. In certain embodiments, R^(B) is C₁₃alkyl. In some embodiments, R^(B) is n-tridecyl. In certain embodiments,R^(B) is C₁₄ alkyl. In some embodiments, R^(B) is n-tetradecyl.

In certain embodiments, p is 1. In certain embodiments, p is 2.

As defined generally above, each R¹ is, independently, fluoro or C₁₋₆alkyl optionally substituted with one or more fluorine radicals. In someembodiments, R¹ is fluoro. In some embodiments, R¹ is C₁₋₆ alkyloptionally substituted with one or more fluorine radicals. In someembodiments, R¹ is unsubstituted C₁₋₆ alkyl. In some embodiments, R¹ ismethyl or ethyl. In some embodiments, R¹ is —CF₃.

In some embodiments, j is 0. In some embodiments, j is 1. In someembodiments, j is 2. In some embodiments, j is 3. In some embodiments, jis 4.

In certain embodiments, a lipidoid of the present invention is of theFormula (III-a):

or a salt thereof,

-   wherein p, R¹, j, and R are as defined above and described herein,    and

each w is, independently, 1, 2, or 3.

In certain embodiments, w is 1. In certain embodiments, w is 2. Incertain embodiments, w is 3.

In certain embodiments, a lipidoid of the present invention is of theFormula (III-b):

or a salt thereof, wherein R¹, j, w, and R are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (III-c):

wherein w and R are as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (III-d):

wherein w, R¹, and R are as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (III-e):

wherein w, R¹, and R are as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theformula:

wherein R is as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (IV):

or a salt thereof,

-   wherein

each R is, independently, hydrogen or —CH₂CH₂C(═O)OR^(B);

each R^(B) is, independently, C₁₀₋₁₄ alkyl, wherein R^(B) is optionallysubstituted with one or more fluorine radicals;

x is 1 or 2; and

y is 1 or 2.

As defined generally above, each R is, independently, hydrogen or—CH₂CH₂C(═O)OR^(B). In some embodiments, at least one R group is—CH₂CH₂C(═O)OR^(B). In some embodiments, at least two R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least three R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least four R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, all R groups are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each R^(B) is, independently, C₁₀₋₁₄ alkyl,wherein R^(B) is optionally substituted with one or more fluorineradicals. In some embodiments, R^(B) is substituted with one or morefluorine radicals. For example, in some embodiments, R^(B) may besubstituted with one fluoro, or in other embodiments, may beperfluorinated. In other embodiments, R^(B) is unsubstituted. In someembodiments, all R^(B) groups are the same. In certain embodiments,R^(B) is C₁₀ alkyl. In some embodiments, R^(B) is n-decyl. In certainembodiments, R^(B) is C₁₁ alkyl. In some embodiments, R^(B) isn-undecyl. In certain embodiments, R^(B) is C₁₂ alkyl. In someembodiments, R^(B) is n-dodecyl. In certain embodiments, R^(B) is C₁₃alkyl. In some embodiments, R^(B) is n-tridecyl. In certain embodiments,R^(B) is C₁₄ alkyl. In some embodiments, R^(B) is n-tetradecyl.

In some embodiments, x is 1. In some embodiments, x is 2. In someembodiments, y is 1. In some embodiments, y is 2. In some embodiments, xis 1 and y is 1. In some embodiments, x is 2 and y is 2.

In certain embodiments, a lipidoid of the present invention is of theFormula (IV-a):

or a salt thereof, wherein R is as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theFormula (V):

or a salt thereof, wherein

each L is, independently, branched or unbranched C₁₋₆ alkylene, whereinL is optionally substituted with one or more fluorine radicals;

each R² is, independently, halo, C₁₋₆ aliphatic optionally substitutedwith one or more fluorine radicals, —OR^(x), —N(R^(y))₂, —SR^(x), —CN,—C(═Z)R^(y), —C(═Z)ZR^(y), or —ZC(═Z)ZR^(y);

Z is O or N;

each R^(x) is, independently, C₁₋₆ aliphatic;

each R^(y) is, independently, hydrogen or C₁₋₆ aliphatic;

g is 0, 1, 2, 3, or 4;

each R is independently hydrogen or —CH₂CH₂C(═O)OR^(B); and

each R^(B) is independently C₁₀₋₁₄ alkyl, wherein R^(B) is optionallysubstituted with one or more fluorine radicals.

As defined generally above, each L is, independently, branched orunbranched C₁₋₆ alkylene, wherein L is optionally substituted with oneor more fluorine radicals. In some embodiments, L is substituted withone or more fluorine radicals. In other embodiments, L is unsubstituted.In some embodiments, L is branched. In other embodiments, L isunbranched. In certain embodiments, L is C₁₋₄ alkylene. In certainembodiments, L is methylene, ethylene, or propylene.

As defined generally above, each R is, independently, hydrogen or—CH₂CH₂C(═O)OR^(B). In some embodiments, at least one R group is—CH₂CH₂C(═O)OR^(B). In some embodiments, at least two R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least three R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least four R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, all R groups are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each R^(B) is, independently, C₁₀₋₁₄ alkyl,wherein R^(B) is optionally substituted with one or more fluorineradicals. In some embodiments, R^(B) is substituted with one or morefluorine radicals. For example, in some embodiments, R^(B) may besubstituted with one fluoro, or in other embodiments, may beperfluorinated. In other embodiments, R^(B) is unsubstituted. In someembodiments, all R^(B) groups are the same. In certain embodiments,R^(B) is C₁₀ alkyl. In some embodiments, R^(B) is n-decyl. In certainembodiments, R^(B) is C₁₁ alkyl. In some embodiments, R^(B) isn-undecyl. In certain embodiments, R^(B) is C₁₂ alkyl. In someembodiments, R^(B) is n-dodecyl. In certain embodiments, R^(B) is C₁₃alkyl. In some embodiments, R^(B) is n-tridecyl. In certain embodiments,R^(B) is C₁₄ alkyl. In some embodiments, R^(B) is n-tetradecyl.

As defined generally above, each R² is, independently, halo, C₁₋₆aliphatic optionally substituted with one or more fluorine radicals,—OR^(x), —N(R^(y))₂, —SR^(x), —CN, —C(═Z)R^(y), —C(═Z)ZR^(y),—ZC(═Z)ZR^(y); wherein Z is O or N; each R^(x) is, independently, C₁₋₆aliphatic; and each R^(y) is, independently, hydrogen or C₁₋₆ aliphatic.In some embodiments, R² is halo. In some embodiments, R² is fluoro. Insome embodiments, R² is C₁₋₆ aliphatic optionally substituted with oneor more fluorine radicals. In some embodiments, R² is C₁₋₆ alkyl.

In some embodiments, g is 0. In some embodiments, g is 1. In someembodiments, g is 2. In some embodiments, g is 3. In some embodiments, gis 4.

In certain embodiments, a lipidoid of the present invention is ofFormula (V-a):

or a salt thereof, wherein L, R², g, and R are as defined above anddescribed herein.

In certain embodiments, a lipidoid of the present invention is ofFormula (V-b):

or a salt thereof, wherein L and R are as defined above and describedherein.

In certain embodiments, a lipidoid of the present invention is ofFormula (V-c):

or a salt thereof, wherein L and R are as defined above and describedherein.

In certain embodiments, a lipidoid of the present invention is ofFormula (V-d):

or a salt thereof, wherein R is as defined above and described herein.

In certain embodiments, a lipidoid of the present invention is of theformula:

or a salt thereof, wherein

each R^(A) is, independently, branched or unbranched C₁₋₆ alkyl, C₃₋₇cycloalkyl, or branched or unbranched C₄₋₁₂ cycloalkylalkyl, whereinR^(A) is optionally substituted with one or more fluorine radicals;

each R is, independently, hydrogen or —CH₂CH₂C(═O)OR^(B); and

each R^(B) is, independently, C₁₀₋₁₄ alkyl, wherein R^(B) is optionallysubstituted with one or more fluorine radicals.

As defined generally above, each R^(A) is, independently, branched orunbranched C₁₋₆ alkyl, C₃₋₇ cycloalkyl, or branched or unbranched C₄₋₁₂cycloalkylalkyl, wherein R^(A) is optionally substituted with one ormore fluorine radicals. In some embodiments, R^(A) is substituted withone or more fluorine radicals. For example, when R^(A) is methyl, it maybe substituted with one, two, or three fluorine radicals to give —CH₂F,—CHF₂, or —CF₃. In other embodiments, R^(A) is unsubstituted. In someembodiments, all R^(A) groups are the same. In other embodiments, theR^(A) groups are different. In some embodiments, R^(A) is branched orunbranched C₁₋₆ alkyl. In certain embodiments, R^(A) is branched C₁₋₆alkyl. In certain embodiments, R^(A) is unbranched C₁₋₆ alkyl. Incertain embodiments, R^(A) is C₁₋₃ alkyl. In certain embodiments, R^(A)is methyl, ethyl, or propyl. In certain embodiments, R^(A) is C₃₋₇cycloalkyl. In certain embodiments, R^(A) is cyclohexyl. In certainembodiments, R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl. Incertain embodiments, R^(A) is cycloheptyl. In some embodiments, R^(A) isbranched or unbranched C₄₋₁₂ cycloalkylalkyl.

As defined generally above, each R is, independently, hydrogen or—CH₂CH₂C(═O)OR^(B). In some embodiments, at least one R group is—CH₂CH₂C(═O)OR^(B). In some embodiments, at least two R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least three R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, at least four R groups are—CH₂CH₂C(═O)OR^(B). In some embodiments, all R groups are—CH₂CH₂C(═O)OR^(B).

As defined generally above, each R^(B) is, independently, C₁₀₋₁₄ alkyl,wherein R^(B) is optionally substituted with one or more fluorineradicals. In some embodiments, R^(B) is substituted with one or morefluorine radicals. For example, in some embodiments, R^(B) may besubstituted with one fluoro, or in other embodiments, may beperfluorinated. In other embodiments, R^(B) is unsubstituted. In someembodiments, all R^(B) groups are the same. In certain embodiments,R^(B) is C₁₀ alkyl. In some embodiments, R^(B) is n-decyl. In certainembodiments, R^(B) is C₁₁ alkyl. In some embodiments, R^(B) isn-undecyl. In certain embodiments, R^(B) is C₁₂ alkyl. In someembodiments, R^(B) is n-dodecyl. In certain embodiments, R^(B) is C₁₃alkyl. In some embodiments, R^(B) is n-tridecyl. In certain embodiments,R^(B) is C₁₄ alkyl. In some embodiments, R^(B) is n-tetradecyl.

In certain embodiments, a lipidoid of the present invention is of theformula:

or a salt thereof, wherein R is as defined above and described herein.

In some embodiments, a lipidoid of the present invention is a compoundresulting from a Michael addition between any one of the amines shown inFIG. 1 or FIG. 2 and an acrylate shown in FIG. 1. In certainembodiments, the number of equivalents of acrylate can be controlled toobtain the desired number of lipid tails on the inventive lipidoid.

In certain embodiments, an inventive lipidoid is prepared by reactingamine 113 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound113O₁₀, 113O₁₁, 113O₁₂, 113O₁₃, or 113O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 11 or 12.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 123 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound123O₁₀, 123O₁₁, 123O₁₂, 123O₁₃, or 123O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 154 with acrylate O₁₀, O_(n), O₁₂, O₁₃, or O₁₄ to form compound154O₁₀, 154O₁₁, 154O₁₂, 154O₁₃, or 154O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 191 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound191O₁₀, 191O₁₁, 191O₁₂, 191O₁₃, or 191O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 10, 11, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 192 with acrylate O₁₀, O_(n), O₁₂, O₁₃, or O₁₄ to form compound192O₁₀, 192O₁₁, 192O₁₂, 192O₁₃, or 192O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 193 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound193O₁₀, 193O₁₁, 193O₁₂, 193O₁₃, or 193O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 195 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound195O₁₀, 195O₁₁, 195O₁₂, 195O₁₃, or 195O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 196 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound196O₁₀, 196O₁₁, 196O₁₂, 196O₁₃, or 196O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 200 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound200O₁₀, 200O₁₁, 200O₁₂, 200O₁₃, or 200O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 205 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound205O₁₀, 205O₁₁, 205O₁₂, 205O₁₃, or 205O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 217 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound217O₁₀, 217O₁₁, 217O₁₂, 217O₁₃, or 217O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 11 or 12.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 218 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound218O₁₀, 218O₁₁, 218O₁₂, 218O₁₃, or 218O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 232 with acrylate O₁₀, O_(n), O₁₂, O₁₃, or O₁₄ to form compound232O₁₀, 232O₁₁, 232O₁₂, 232O₁₃, or 232O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 235 with acrylate O₁₀, O_(n), O₁₂, O₁₃, or O₁₄ to form compound235O₁₀, 235O₁₁, 235O₁₂, 235O₁₃, or 235O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 302 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound302O₁₀, 302O₁₁, 302O₁₂, 302O₁₃, or 302O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 12 or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 303 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound303O₁₀, 303O₁₁, 303O₁₂, 303O₁₃, or 303O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 304 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound304O₁₀, 304O₁₁, 304O₁₂, 304O₁₃, or 304O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 11 or 12.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 305 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound305O₁₀, 305O₁₁, 305O₁₂, 305O₁₃, or 305O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 306 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound306O₁₀, 306O₁₁, 306O₁₂, 306O₁₃, or 306O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 313 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound313O₁₀, 313O₁₁, 313O₁₂, 313O₁₃, or 313O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, or 12.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 315 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound315O₁₀, 315O₁₁, 315O₁₂, 315O₁₃, or 315O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 347 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound347O₁₀, 347O₁₁, 347O₁₂, 347O₁₃, or 347O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 11 or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 366 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound366O₁₀, 366O₁₁, 366O₁₂, 366O₁₃, or 366O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 10 or 11.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 371 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound371O₁₀, 371O₁₁, 371O₁₂, 371O₁₃, or 371O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 500 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound500O₁₀, 500O₁₁, 500O₁₂, 500O₁₃, or 500O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 501 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound501O₁₀, 501O₁₁, 501O₁₂, 501O₁₃, or 501O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 502 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound502O₁₀, 502O₁₁, 502O₁₂, 502O₁₃, or 502O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 503 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound503O₁₀, 503O₁₁, 503O₁₂, 503O₁₃, or 503O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 504 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound504O₁₀, 504O₁₁, 504O₁₂, 504O₁₃, or 504O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 505 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound505O₁₀, 505O₁₁, 505O₁₂, 505O₁₃, or 505O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 506 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound506O₁₀, 506O₁₁, 506O₁₂, 506O₁₃, or 506O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 507 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound507O₁₀, 507O₁₁, 507O₁₂, 507O₁₃, or 507O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 508 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound508O₁₀, 508O₁₁, 508O₁₂, 508O₁₃, or 508O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 509 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound509O₁₀, 509O₁₁, 509O₁₂, 509O₁₃, or 509O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 510 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound510O₁₀, 510O₁₁, 510O₁₂, 510O₁₃, or 510O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 511 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound511O₁₀, 511O₁₁, 511O₁₂, 511O₁₃, or 511O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 512 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound512O₁₀, 512O₁₁, 512O₁₂, 512O₁₃, or 512O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 513 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound513O₁₀, 513O₁₁, 513O₁₂, 513O₁₃, or 513O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 514 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound514O₁₀, 514O₁₁, 514O₁₂, 514O₁₃, or 514O₁₄. In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

In certain embodiments, an inventive lipidoid is prepared by reactingamine 515 with acrylate O₁₀, O₁₁, O₁₂, O₁₃, or O₁₄ to form compound515O₁₀, 515O₁₁, 515O₁₂, 515O₁₃, or 515O₁₄ In certain embodiments, aninventive lipidoid is of one of the formulae below:

wherein z is 9, 10, 11, 12, or 13.

In some embodiments, the present invention provides a composition of oneor more of the above lipidoids. In certain embodiments, an inventivelipidoid is of the formula:

Synthesis of Lipids

Lipidoids described herein may be prepared by any method known in theart. In certain embodiments, inventive lipidoids are prepared via theconjugate addition of primary or secondary amines to acrylates. Suchsyntheses are described in detail in U.S. Publication No. 2011/0009641,incorporated herein by reference. In certain embodiments, inventivelipidoids are prepared from commercially available starting materials,such acrylates and amines. In other embodiments, inventive lipidoids areprepared from easily and/or inexpensively prepared starting materials.As would be appreciated by one of skill in the art, the lipidoidsdescribed herein can be prepared by total synthesis starting fromcommercially available starting materials. A particular lipidoid may bethe desired final product of the synthesis, or a mixture of lipidoidsmay be the desired final product.

Polynucleotide Complexes

The ability of cationic compounds to interact with negatively chargedpolynucleotides through electrostatic interactions is well known.Cationic lipids such as Lipofectamine have been prepared and studied fortheir ability to complex and transfect polynucleotides. The interactionof the lipid with the polynucleotide is thought to at least partiallyprevent the degradation of the polynucleotide. By neutralizing thecharge on the backbone of the polynucleotide, the neutral orslightly-positively-charged complex is also able to more easily passthrough the hydrophobic membranes (e.g., cytoplasmic, lysosomal,endosomal, nuclear) of the cell. In certain embodiments, the complex isslightly positively charged. In certain embodiments, the complex has apositive ζ-potential. In certain embodiments, the ζ-potential is between+1 and +30.

In certain embodiments, lipidoids of the present invention possesstertiary amines. Although these amines are hindered, they are availableto interact with a polynucleotide (e.g., DNA, RNA, synthetic analogs ofDNA and/or RNA, DNA/RNA hydrids, etc.). In certain embodiments,polynucleotides or derivatives thereof are contacted with the inventivelipidoids under conditions suitable to form polynucleotide/lipidoidcomplexes. In certain embodiments, the lipidoid is at least partiallyprotonated so as to form a complex with the negatively chargedpolynucleotide. In certain embodiments, the polynucleotide/lipidoidcomplexes form nanoparticles that are useful in the delivery ofpolynucleotides to cells. In certain embodiments, multiple lipidoidmolecules may be associated with a polynucleotide molecule. The complexmay include 1-100 lipidoid molecules, 1-1000 lipidoid molecules, 10-1000lipidoid molecules, or 100-10,000 lipidoid molecules. In certainembodiments, the complex may form a nanoparticle. In certainembodiments, the diameter of the nanoparticles ranges from 10-500 nm,from 10-1200 nm, or from 50-150 nm. In certain embodiments,nanoparticles may be associated with a targeting agent as describedbelow.

Polynucleotide

A polynucleotide to be complexed, encapsulated by the inventivelipidoids, or included in a composition with the inventive lipidoids maybe any nucleic acid including but not limited to RNA and DNA. In certainembodiments, the polynucleotide is DNA. In other embodiments, thepolynucleotide is RNA. In certain embodiments, the polynucleotide is ansiRNA. In certain embodiments, the polynucleotide is an shRNA. Incertain embodiments, the polynucleotide is an mRNA. In certainembodiments, the polynucleotide is a dsRNA. In certain embodiments, thepolynucleotide is an miRNA. In certain embodiments, the polynucleotideis an antisense RNA. The polynucleotides may be of any size or sequence,and they may be single- or double-stranded. In certain embodiments, thepolynucleotide is greater than 100 base pairs long. In certain otherembodiments, the polynucleotide is greater than 1000 base pairs long andmay be greater than 10,000 base pairs long. In certain embodiments, thepolynucleotide is purified and substantially pure. In certainembodiments, the polynucleotide is greater than 50% pure, greater than75% pure, or greater than 95% pure. The polynucleotide may be providedby any means known in the art. In certain preferred embodiments, thepolynucleotide has been engineered using recombinant techniques (for amore detailed description of these techniques, please see Ausubel et al.Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NewYork, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press:1989); each of which is incorporated herein by reference). Thepolynucleotide may also be obtained from natural sources and purifiedfrom contaminating components found normally in nature. Thepolynucleotide may also be chemically synthesized in a laboratory. Incertain embodiments, the polynucleotide is synthesized using standardsolid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain embodiments, these modifications lead to increased stability ofthe polynucleotide. Modifications include methylation, phosphorylation,end-capping, etc.

Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, 3′-azido-2′,3′-dideoxyribose, 2′,3′-dideoxyribose,arabinose (the 2′-epimer of ribose), acyclic sugars, and hexoses. Thenucleosides may be strung together by linkages other than thephosphodiester linkage found in naturally occurring DNA and RNA.Modified linkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Combinations of the various modificationsmay be used in a single polynucleotide. These modified polynucleotidesmay be provided by any means known in the art; however, as will beappreciated by those of skill in this art, the modified polynucleotidesare preferably prepared using synthetic chemistry in vitro. Thepolynucleotides to be delivered may be in any form. For example, thepolynucleotide may be a circular plasmid, a linearized plasmid, acosmid, a viral genome, a modified viral genome, an artificialchromosome, etc.

The polynucleotide may be of any sequence. In certain preferredembodiments, the polynucleotide encodes a protein or peptide. Theencoded proteins may be enzymes, structural proteins, receptors, solublereceptors, ion channels, pharmaceutically active proteins, cytokines,interleukins, antibodies, antibody fragments, antigens, coagulationfactors, albumin, growth factors, hormones, insulin, etc. Thepolynucleotide may also comprise regulatory regions to control theexpression of a gene. These regulatory regions may include, but are notlimited to, promoters, enhancer elements, repressor elements, TATA box,ribosomal binding sites, stop site for transcription, etc. In otherparticularly preferred embodiments, the polynucleotide is not intendedto encode a protein. For example, the polynucleotide may be used to fixan error in the genome of the cell being transfected.

The polynucleotide may also be provided as an antisense agent or RNAinterference (RNAi) (Fire et al. Nature 391:806-811, 1998; incorporatedherein by reference). Antisense therapy is meant to include, e.g.,administration or in situ provision of single- or double-strandedoligonucleotides or their derivatives which specifically hybridize,e.g., bind, under cellular conditions, with cellular mRNA and/or genomicDNA, or mutants thereof, so as to inhibit expression of the encodedprotein, e.g., by inhibiting transcription and/or translation (Crooke“Molecular mechanisms of action of antisense drugs” Biochim. Biophys.Acta 1489(1):31-44, 1999; Crooke “Evaluating the mechanism of action ofantiproliferative antisense drugs” Antisense Nucleic Acid Drug Dev.10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes313-314, 1999; each of which is incorporated herein by reference). Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix (i.e., triple helixformation) (Chan et al. J. Mol. Med. 75(4):267-282, 1997; incorporatedherein by reference).

In certain embodiments, the polynucleotide to be delivered comprises asequence encoding an antigenic peptide or protein. Nanoparticlescontaining these polynucleotides can be delivered to an individual toinduce an immunologic response sufficient to decrease the chance of asubsequent infection and/or lessen the symptoms associated with such aninfection. The polynucleotide of these vaccines may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. A large number of adjuvantcompounds are known; a useful compendium of many such compounds isprepared by the National Institutes of Health and can be found on theinternet (http:/www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf,incorporated herein by reference; see also Allison Dev. Biol. Stand.92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; andPhillips et al. Vaccine 10:151-158, 1992, each of which is incorporatedherein by reference).

An antigenic protein or peptides encoded by a polynucleotide may bederived from such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; from suchviruses as smallpox, influenza A and B, respiratory syncytial virus,parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2,cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, hepatitis A, B, C, D, and E virus, and the like; and from suchfungal, protozoan, and parasitic organisms such as Cryptococcusneoformans, Histoplasma capsulatum, Candida albicans, Candidatropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi,Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica,Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and thelike.

Microparticles and Nanoparticles

The lipidoids of the present invention may also be used to form drugdelivery devices. Inventive lipidoids may be used to encapsulate agentsincluding polynucleotides, small molecules, proteins, peptides, metals,organometallic compounds, etc. Lipidoids described herein have severalproperties that make them particularly suitable in the preparation ofdrug delivery devices. These include 1) the ability of the lipid tocomplex and “protect” labile agents; 2) the ability to buffer the pH inthe endosome; 3) the ability to act as a “proton sponge” and causeendosomolysis; and 4) the ability to neutralize the charge on negativelycharged agents. In certain embodiments, the diameter of the particlesrange from between 1 micrometer to 1,000 micrometers. In certainembodiments, the diameter of the particles range from between from 1micrometer to 100 micrometers. In certain embodiments, the diameter ofthe particles range from between from 1 micrometer to 10 micrometers. Incertain embodiments, the diameter of the particles range from betweenfrom 10 micrometer to 100 micrometers. In certain embodiments, thediameter of the particles range from between from 100 micrometer to1,000 micrometers. In certain embodiments, the particles range from 1-5micrometers. In certain embodiments, the diameter of the particles rangefrom between 1 nm to 1,000 nm. In certain embodiments, the diameter ofthe particles range from between from 1 nm to 100 nm. In certainembodiments, the diameter of the particles range from between from 1 nmto 10 nm. In certain embodiments, the diameter of the particles rangefrom between from 10 nm to 100 nm. In certain embodiments, the diameterof the particles range from between from 100 nm to 1,000 nm. In certainembodiments, the diameter of the particles range from between from 20 nmto 2,000 nm. In certain embodiments, the particles range from 1-5 nm. Incertain embodiments, the diameter of the particles range from between 1pm to 1,000 pm. In certain embodiments, the diameter of the particlesrange from between from 1 pm to 100 pm. In certain embodiments, thediameter of the particles range from between from 1 pm to 10 pm. Incertain embodiments, the diameter of the particles range from betweenfrom 10 pm to 100 pm. In certain embodiments, the diameter of theparticles range from between from 100 pm to 1,000 pm. In certainembodiments, the particles range from 1-5 pm.

The inventive particles may be prepared using any method known in thisart. These include, but are not limited to, spray drying, single anddouble emulsion solvent evaporation, solvent extraction, phaseseparation, simple and complex coacervation, and other methods wellknown to those of ordinary skill in the art. In certain embodiments,methods of preparing the particles are the double emulsion process andspray drying. The conditions used in preparing the particles may bealtered to yield particles of a desired size or property (e.g.,hydrophobicity, hydrophilicity, external morphology, “stickiness”,shape, etc.). The method of preparing the particle and the conditions(e.g., solvent, temperature, concentration, air flow rate, etc.) usedmay also depend on the agent being encapsulated and/or the compositionof the matrix. Methods developed for making particles for delivery ofencapsulated agents are described in the literature (for example, pleasesee Doubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine andPharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J.Controlled Release 5:13-22, 1987; Mathiowitz et al., Reactive Polymers6:275-283, 1987; Mathiowitz et al., J. Appl. Polymer Sci. 35:755-774,1988; each of which is incorporated herein by reference).

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve. The particle may also be coated. In certain embodiments,the particles are coated with a targeting agent. In other embodiments,the particles are coated to achieve desirable surface properties (e.g.,a particular charge).

In certain embodiments, the present invention provides a nanoparticlecomprising an inventive lipidoid and one or more agents to be delivered.In certain embodiments, the agent is a polynucleotide, drug, protein orpeptide, small molecule, or gas. In certain embodiments, the agent isRNA (e.g. mRNA, RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA). Incertain embodiments, the nanoparticle further comprises cholesterol or aderivative thereof, such as3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-cholesterol). In certain embodiments, the nanoparticle furthercomprises a PEG-based material. In certain embodiments, the PEG-basedmaterial is PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxyPEG. For instance, in certain embodiments, the PEG-based material is C14PEG2000 DMG, C15 PEG2000 DMG, C16 PEG2000 DMG, C18 PEG2000 DMG, C14 PEG2000 ceramide, C15 PEG2000 ceramide, C16 PEG2000 ceramide, C18 PEG2000ceramide, C14 PEG2000 PE, C15 PEG2000 PE, C16 PEG2000 PE, C18 PEG2000PE, C14 PEG350 PE, C14 PEG5000 PE, poloxamer F-127, poloxamer F-68,poloxamer L-64, or DSPE carboxy PEG. In certain embodiments, thenanoparticle further comprises a lipid. For example, in certainembodiments, the nanoparticle further comprises DSPC, DOPC, or DOPE. Incertain embodiments, the nanoparticle comprises a lipidoid, an agent(e.g., RNA), a lipid, cholesterol or a derivative thereof, and aPEG-based material.

Micelles, Liposomes, and Lipoplexes

Lipidoids described herein may also be used to prepare micelles orliposomes. In addition, any agent may be included in a micelle orliposome. Micelles and liposomes are particularly useful in deliveringhydrophobic agents such as hydrophobic small molecules. When the micelleor liposome is complexed with (e.g., encapsulates or covers) apolynucleotide it is referred to as a “lipoplex.” Many techniques forpreparing micelles, liposomes, and lipoplexes are known in the art, andany method may be used with the inventive lipidoids to make micelles andliposomes.

In certain embodiments, liposomes (lipid vesicles) are formed throughspontaneous assembly. In other embodiments, liposomes are formed whenthin lipid films or lipid cakes are hydrated and stacks of lipidcrystalline bilayers become fluid and swell. The hydrated lipid sheetsdetach during agitation and self-close to form large, multilamellarvesicles (LMV). This prevents interaction of water with the hydrocarboncore of the bilayers at the edges. Once these particles have formed,reducing the size of the particle can be modified through input of sonicenergy (sonication) or mechanical energy (extrusion). See Walde, P.“Preparation of Vesicles (Liposomes)” In Encylopedia of Nanoscience andNanotechnology; Nalwa, H. S. Ed. American Scientific Publishers LosAngeles, 2004; Vol. 9, pp. 43-79; Szoka et al. “Comparative Propertiesand Methods of Preparation of Lipid Vesicles (Liposomes)” Ann. Rev.Biophys. Bioeng. 9:467-508, 1980; each of which is incorporated herein.The preparation of lipsomes involves preparing the lipid for hydration,hydrating the lipid with agitation, and sizing the vesicles to achieve ahomogenous distribution of liposomes. Lipids are first dissolved in anorganic solvent to assure a homogeneous mixture of lipids. The solventis then removed to form a lipid film. This film is thoroughly dried toremove residual organic solvent by placing the vial or flask on avaccuum pump overnight. Hydration of the lipid film/cake is accomplishedby adding an aqueous medium to the container of dry lipid and agitatingthe mixture. Disruption of LMV suspensions using sonic energy typicallyproduces small unilamellar vesicles (SUV) with diameters in the range of15-50 nm. Lipid extrusion is a technique in which a lipid suspension isforced through a polycarbonate filter with a defined pore size to yieldparticles having a diameter near the pore size of the filter used.Extrusion through filters with 100 nm pores typically yields large,unilamellar vesicles (LUV) with a mean diameter of 120-140 nm.

In certain embodiments, liposomes are formed comprising an inventivelipid, a PEG-based material, cholesterol or a derivative thereof, and apolynucleotide. In certain embodiments, the polynucleotide is an RNAmolecule (e.g., an siRNA). In other embodiments, the polynucleotide is aDNA molecule. In certain embodiments, the amount of lipidoid in theliposome ranges from 30-80 mol %, 40-70 mol %, or 60-70 mol %. Incertain embodiments, the liposome comprises a PEG-based material. Incertain embodiments, the amount of PEG-based material in the liposomesranges from 5-20 mol %, 10-15 mol %, or 10 mol %. In certainembodiments, the liposome comprises cholesterol or a derivative thereof.In certain embodiments, the amount of cholesterol in the liposome rangesfrom 5-25 mol %, 10-20 mol %, or 15 mol %. In certain embodiments, theamount of cholesterol in the liposome is approximately 20 mol %. Theseliposomes may be prepared using any method known in the art. In certainembodiments (e.g., liposomes containing RNAi molecules), the liposomesare prepared by lipid extrusion.

Certain lipidoids can spontaneously self assemble around certainmolecules, such as DNA and RNA, to form liposomes. In some embodiments,the application is the delivery of polynucleotides. Use of theselipidoids allows for simple assembly of liposomes without the need foradditional steps or devices such as an extruder.

The following scientific papers described other methods for preparingliposomes and micelles: Narang et al. “Cationic Lipids with IncreasedDNA Binding Affinity for Nonviral Gene Transfer in Dividing andNondividing Cells” Bioconjugate Chem. 16:156-68, 2005; Hofland et al.“Formation of stable cationic lipid/DNA complexes for gene transfer”Proc. Natl. Acad. Sci. USA 93:7305-7309, July 1996; Byk et al.“Synthesis, Activity, and Structure—Activity Relationship Studies ofNovel Cationic Lipids for DNA Transfer” J. Med. Chem. 41(2):224-235,1998; Wu et al. “Cationic Lipid Polymerization as a Novel Approach forConstructing New DNA Delivery Agents” Bioconjugate Chem. 12:251-57,2001; Lukyanov et al. “Micelles from lipid derivatives of water-solublepolymers as delivery systems for poorly soluble drugs” Advanced DrugDelivery Reviews 56:1273-1289, 2004; Tranchant et al. “Physicochemicaloptimisation of plasmid delivery by cationic lipids” J. Gene Med.6:S24-S35, 2004; van Balen et al. “Liposome/Water Lipophilicity:Methods, Information Content, and Pharmaceutical Applications” MedicinalResearch Rev. 24(3):299-324, 2004; each of which is incorporated hereinby reference.Agent

The agents to be delivered by the system of the present invention may betherapeutic, diagnostic, or prophylactic agents. Any chemical compoundto be administered to an individual may be delivered using the inventiveinventive complexes, picoparticles, nanoparticles, microparticles,micelles, or liposomes. The agent may be a small molecule,organometallic compound, nucleic acid, protein, peptide, polynucleotide,targeting agent, an isotopically labeled chemical compound, drug,vaccine, immunological agent, etc. In certain embodiments, the agentsare organic compounds with pharmaceutical activity. In anotherembodiment of the invention, the agent is a clinically used drug. In aparticularly preferred embodiment, the drug is an antibiotic,chemotherapeutic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, etc.

In certain embodiments, the agent to be delivered may be a mixture ofagents.

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodine-based materials. Prophylactic agents include, but are not limitedto, antibiotics, nutritional supplements, and vaccines. Vaccines maycomprise isolated proteins or peptides, inactivated organisms andviruses, dead organisms and viruses, genetically altered organisms orviruses, and cell extracts. Prophylactic agents may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. Prophylactic agents includeantigens of such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

Targeting Agents

The inventive lipidoids, and the complexes, liposomes, micelles,microparticles, picoparticles and nanoparticles prepared therefrom, maybe modified to include targeting agents since it is often desirable totarget a particular cell, collection of cells, or tissue. A variety oftargeting agents that direct pharmaceutical compositions to particularcells are known in the art (see, for example, Cotten et al. MethodsEnzym. 217:618, 1993; incorporated herein by reference). The targetingagents may be included throughout the particle or may be only on thesurface. The targeting agent may be a protein, peptide, carbohydrate,glycoprotein, lipid, small molecule, etc. The targeting agent may beused to target specific cells or tissues or may be used to promoteendocytosis or phagocytosis of the particle. Examples of targetingagents include, but are not limited to, antibodies, fragments ofantibodies, low-density lipoproteins (LDLs), transferrin,asialycoproteins, gp120 envelope protein of the human immunodeficiencyvirus (HIV), carbohydrates, receptor ligands, sialic acid, etc. If thetargeting agent is included throughout the particle, the targeting agentmay be included in the mixture that is used to form the particles. Ifthe targeting agent is only on the surface, the targeting agent may beassociated with (i.e., by covalent, hydrophobic, hydrogen bonding, vander Waals, or other interactions) the formed particles using standardchemical techniques.

Compositions

In certain embodiments, an inventive lipidoid is a component of acomposition which may be useful in a variety of medical and non-medicalapplications. For example, pharmaceutical compositions comprising aninventive lipidoid may be useful in the delivery of an effective amountof an agent to a subject in need thereof. Nutraceutical compositionscomprising an inventive lipidoid may be useful in the delivery of aneffective amount of a nutraceutical, e.g., a dietary supplement, to asubject in need thereof. Cosmetic compositions comprising an inventivelipidoid may be formulated as a cream, ointment, balm, paste, film, orliquid, etc., and may be useful in the application of make-up, hairproducts, and materials useful for personal hygiene, etc.

In certain embodiments, the composition comprises one or more lipidoidsof the present invention. “One or more lipidoids” refers to one or moredifferent types of lipidoids included in the composition, andencompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types oflipidoids.

In certain embodiments, the inventive lipidoids are useful incompositions, either for delivery of an effective amount of an agent toa subject in need thereof (e.g., a pharmaceutical composition, acosmetic composition) or for use as an excipient. For example, cosmeticcompositions may further use the inventive lipidoids as excipientsrather than as a delivery system encapsulating an agent to be delivered.In certain embodiments, the composition is a pharmaceutical composition.In certain embodiments, the composition is a cosmetic composition.

In certain embodiments, the composition further comprises an agent, asdescribed herein. For example, in certain embodiments, the agent is asmall molecule, organometallic compound, nucleic acid, protein, peptide,polynucleotide, metal, targeting agent, an isotopically labeled chemicalcompound, drug, vaccine, or immunological agent. In certain embodiments,the agent is a polynucleotide. In certain embodiments, thepolynucleotide is DNA or RNA. In certain embodiments, the RNA is mRNA,RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.

In certain embodiments, the polynucleotide and the one or more lipidoidsare not covalently attached.

In certain embodiments, the one or more lipidoids are in the form of aparticle. In certain embodiments, the particle is a nanoparticle ormicroparticle. In certain embodiments, the one or more conjugatedlipidoids are in the form of liposomes or micelles. It is understoodthat, in certain embodiments, these lipidoids self-assemble to provide aparticle, micelle or liposome. In certain embodiments, the particle,liposome, or micelle encapsulates an agent. The agent to be delivered bythe particles, liposomes, or micelles may be in the form of a gas,liquid, or solid. The inventive lipidoids may be combined with polymers(synthetic or natural), surfactants, cholesterol, carbohydrates,proteins, lipids etc. to form the particles. These particles may becombined with an excipient to form pharmaceutical and cosmeticcompositions.

Once the complexes, micelles, liposomes, or particles have beenprepared, they may be combined with one or more excipients to form acomposition that is suitable to administer to animals including humans.

As would be appreciated by one of skill in this art, the excipients maybe chosen based on the route of administration as described below, theagent being delivered, time course of delivery of the agent, etc.

In certain embodiments, provided is a composition comprising aninventive lipidoids and an excipient. As used herein, the term“excipient” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as excipients include, butare not limited to, sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator. The compositions of this invention can be administered tohumans and/or to animals, orally, rectally, parenterally,intracisternally, intravaginally, intranasally, intraperitoneally,topically (as by powders, creams, ointments, or drops), bucally, or asan oral or nasal spray.

Liquid dosage forms for oral administration include emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredients (i.e., microparticles, nanoparticles,liposomes, micelles, polynucleotide/lipid complexes), the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Incertain embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the particles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

Methods of Use

In another aspect, provided are methods of using the inventivelipidoids, e.g., for the treatment of a disease, disorder or conditionfrom which a subject suffers. It is contemplated that the inventivelipidoids will be useful in the treatment of a variety of diseases,disorders or conditions, especially as a system for delivering agentsuseful in the treatment of that particular disease, disorder orcondition.

For example, in one aspect, provided is a method of treating cancercomprising administering to a subject in need thereof an effectiveamount of a lipidoid of the present invention, or salt thereof, or acomposition thereof. In certain embodiments, the method furthercomprises administering an anti-cancer agent. In certain embodiments,the lipidoid encapsulates the anti-cancer agent. In certain embodiments,the lipidoid and the anti-cancer agent form a particle (e.g., ananoparticle, a microparticle, a micelle, a liposome, a lipoplex).

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example mammals (e.g., primates (e.g., cynomolgusmonkeys, rhesus monkeys); commercially relevant mammals such as cattle,pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys), reptiles, amphibians, and fish. In certain embodiments, thenon-human animal is a mammal. The non-human animal may be a male orfemale and at any stage of development. A non-human animal may be atransgenic animal.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from the specified disease, disorder or condition,which reduces the severity of the disease, disorder or condition, orretards or slows the progression of the disease, disorder or condition(“therapeutic treatment”), and also contemplates an action that occursbefore a subject begins to suffer from the specified disease, disorderor condition (“prophylactic treatment”).

In general, the “effective amount” of a compound refers to an amountsufficient to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof a compound of the invention may vary depending on such factors as thedesired biological endpoint, the pharmacokinetics of the compound, thedisease being treated, the mode of administration, and the age, health,and condition of the subject. An effective amount encompassestherapeutic and prophylactic treatment.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment of a disease, disorder orcondition, or to delay or minimize one or more symptoms associated withthe disease, disorder or condition. A therapeutically effective amountof a compound means an amount of therapeutic agent, alone or incombination with other therapies, which provides a therapeutic benefitin the treatment of the disease, disorder or condition. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of disease orcondition, or enhances the therapeutic efficacy of another therapeuticagent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease, disorder or condition, or one or more symptoms associated withthe disease, disorder or condition, or prevent its recurrence. Aprophylactically effective amount of a compound means an amount of atherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the disease,disorder or condition. The term “prophylactically effective amount” canencompass an amount that improves overall prophylaxis or enhances theprophylactic efficacy of another prophylactic agent.

Exemplary cancers include, but are not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

Anti-cancer agents encompass biotherapeutic anti-cancer agents as wellas chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limitedto, interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) andantibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR(tositumomab)). Exemplary chemotherapeutic agents include, but are notlimited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol),LHRH agonists (e.g. goscrclin and leuprolide), anti-androgens (e.g.flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin(BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellinA (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (ABRAXANE),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765,AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523,PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTORinhibitors (e.g., rapamycin, temsirolimus (CCl-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)),oblimersen, gemcitabine, caminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Lipidoid Synthesis

Lipidoids were synthesized through the conjugate addition ofalkyl-acrylates to amines. Amines were purchased from Sigma Aldrich (St.Louis, Mo.), Alfa Aesar, Acros Organics, and CHESS Organics. Acrylateswere purchased from Scientific Polymer Products (Ontario, N.Y.) andHampford Research, Inc. (Stratford, Conn.). Amines were combined withacrylates stoichiometrically in a glass scintillation vial and werestirred at 90° C. for either for 3 days. In vitro experiments wereconducted with crude materials, and in vivo experiments were performedwith lipidoids purified via a Teledyne Isco Chromatography system(Lincoln, Nebr.).

Lipidoid Hydrolysis

To a 25 ml round bottom flask was added 304O₁₃ (0.250 g, 0.263 mmol, 1equiv). For acidic hydrolysis, 10 ml of a solution of 6 N HCl was addedto the flask to afford a cloudy heterogeneous solution. The reaction washeated to reflux to afford a clear, homogeneous solution and was stirredat reflux for 24 hours. For basic hydrolysis, 10 ml of a solution of KOHin EtOH/H₂O (solution=5.61 g KOH in 47.5 ml EtOH w/2.5 ml distilled H₂O)was added to the flask to afford a clear colorless solution. Thereaction was heated to reflux and stirred for 41 h. Both acidic andbasic reactions were cooled to room temperature and TLC analysis showedthe presence of tridecanol (17.5% EtOAC/Hexanes) and the consumption of304O₁₃. Reactions were concentrated to dryness under reduced pressureand diluted with CDCl₃. The basic reaction was filtered to remove excessKOH. Proton NMR analysis was performed in CDCl₃. Proton nuclear magneticresonance spectra were recorded with a Bruker Avance 400 spectrometer,are depicted in parts per million on the δ scale, and are referencedfrom the residual protium in the NMR solvent (CDCl₃: δ 7.26 (CHCl₃).

Formulation of Lipid Nanoparticles

Lipidoids were formulated into nanoparticles for all studies describedin the Examples. Nanoparticles were formed by mixing lipidoids,cholesterol (Sigma Aldrich), DSPC (Avanti Polar Lipids, Alabaster, Ala.)and mPEG2000-DMG (MW 2660, gift from Alnylam Pharamceuticals, Cambridge,Mass.) at a molar ratio of 38.5:50:(11.5-X): X in a solution of 90%ethanol and 10% 10 mM sodium citrate (by volume). An siRNA solution wasprepared by diluting siRNA in 10 mM sodium citrate such that the finalweight ratio of total lipid (lipidoid+cholesterol+DSPC+PEG):siRNA was10:1. Equal volumes of lipid solution and siRNA solution were rapidlymixed together using either a microfluidic device (Chen, D. et al. J.Am. Chem. Soc. 134, 120410134818007 (2012)) or by pipet to formnanoparticles. Particles were diluted in phosphate buffered saline (PBS,Invitrogen) and then dialyzed against PBS for 90 minutes in 3500 MWCOcassettes (Pierce/Thermo Scientific, Rockford, Ill.).

In Vitro Transfection of Cell Lines with Lipid Nanoparticles

HeLa cells stably modified to express both firefly and Renillaluciferase were maintained at 37° C. in high glucose Dulbecco's ModifiedEagles Medium without phenol red (Invitrogen, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (FBS, Invitrogen). 12-16 hoursprior to transfection, cells were seeded in white 96-well plates at adensity of 15,000 cells per well. Cells were transfected with a 40 nMconcentration of anti-firefly luciferase siRNA (Dharmacon, Lafayette,Colo.) that had been formulated with lipidoids into nanoparticles.Firefly luciferase silencing was assessed with a Dual-Glo® LuciferaseAssay kit (Promega, Madison, Wis.). Renilla luciferase activity servedas a control. Data for certain lipidoids are shown in Table 1 below.

TABLE 1 In vitro data Relative Amine Lipid tail Luciferase ActivityStdev 98 O10 0.52 0.02 O11 0.58 0.08 O12 0.63 0.06 O13 0.57 0.07 O140.61 0.07 122 O10 0.37 0.05 O11 0.61 0.04 O12 0.68 0.02 O13 0.75 0.03O14 0.73 0.04 123 O10 0.73 0.05 O11 0.62 0.03 O12 0.70 0.03 O13 0.220.02 O14 0.68 0.14 154 O10 0.74 0.04 O11 0.72 0.04 O12 0.33 0.12 O130.87 0.06 O14 0.88 0.07 174 O10 0.40 0.01 O11 0.47 0.07 O12 0.69 0.05O13 0.55 0.02 O14 0.24 0.01 176 O10 0.20 0.03 O11 0.17 0.01 O12 0.900.03 O13 0.79 0.03 O14 0.63 0.03 191 O10 0.71 0.09 O11 0.31 0.05 O120.32 0.03 O13 0.76 0.06 O14 0.34 0.08 192 O10 0.88 0.03 O11 0.49 0.04O12 0.37 0.04 O13 0.80 0.04 O14 0.60 0.06 193 O10 0.55 0.06 O11 0.380.06 O12 0.28 0.03 O13 0.84 0.03 O14 0.55 0.04 195 O10 0.80 0.06 O110.75 0.04 O12 0.43 0.03 O13 0.28 0.02 O14 0.76 0.04 196 O10 0.92 0.03O11 0.76 0.02 O12 0.85 0.09 O13 0.58 0.04 O14 0.38 0.05 200 O10 0.570.03 O11 0.64 0.03 O12 0.41 0.03 O13 0.53 0.05 O14 0.36 0.01 205 O100.67 0.04 O11 0.68 0.03 O12 0.53 0.05 O13 0.72 0.04 O14 0.43 0.02 217O10 NA NA O11 NA NA O12 0.22 0.03 O13 0.33 0.05 O14 NA NA 218 O10 0.910.04 O11 0.90 0.06 O12 0.80 0.03 O13 0.41 0.06 O14 0.57 0.08 232 O100.92 0.05 O11 0.90 0.07 O12 0.83 0.04 O13 0.39 0.05 O14 0.68 0.02 235O10 0.85 0.10 O11 0.85 0.08 O12 0.85 0.11 O13 0.42 0.03 O14 0.72 0.03302 O10 0.64 0.01 O11 0.64 0.03 O12 0.64 0.03 O13 0.34 0.05 O14 0.360.09 303 O10 0.07 0.01 O11 0.78 0.04 O12 0.40 0.07 O13 0.89 0.05 O140.89 0.08 304 O10 0.86 0.07 O11 0.45 0.02 O12 0.28 0.01 O13 0.08 0.01O14 0.53 0.01 306 O10 0.13 0.02 O11 0.14 0.02 O12 0.09 0.01 O13 0.100.02 O14 0.08 0.01 313 O10 0.37 0.02 O11 0.32 0.02 O12 0.21 0.01 O130.23 0.04 O14 0.63 0.05 315 O10 0.98 0.08 O11 0.89 0.09 O12 0.30 0.01O13 0.65 0.03 O14 0.80 0.04 347 O10 0.68 0.04 O11 0.57 0.02 O12 0.170.06 O13 0.55 0.07 O14 0.17 0.06 366 O10 0.50 0.09 O11 0.38 0.08 O120.38 0.04 O13 0.59 0.08 O14 0.55 0.11 371 O10 0.85 0.01 O11 0.40 0.01O12 0.48 0.02 O13 0.76 0.04 O14 0.39 0.01 500 O10 0.14 0.03 O11 0.090.03 O12 0.06 0.01 O13 0.07 0.01 O14 0.02 0.00 501 O10 0.37 0.02 O110.37 0.04 O12 0.22 0.04 O13 0.24 0.03 O14 0.20 0.08 502 O10 0.18 0.03O11 0.12 0.01 O12 0.08 0.01 O13 0.09 0.01 O14 0.08 0.02 503 O10 0.390.05 O11 0.39 0.06 O12 0.33 0.07 O13 0.07 0.00 O14 0.12 0.04In Vivo Gene Silencing

All animal experiments were conducted using institutionally-approvedprotocols. Female C57BL/6 mice (Charles River Laboratories, Wilmington,Mass.) received injections through the lateral tail vein injections ofPBS (negative control), or lipidoid nanoparticles containing eithernon-targeting siRNA (negative control) or anti-Factor VII siRNA dilutedin PBS at a volume of 0.01 ml/g. The sequence of the siFVII, provided byAlnylam Pharmaceuticals, was:

(SEQ ID NO.: 1) sense: 5′-GGAucAucucAAGucuuAcT*T-3′ (SEQ ID NO.: 2)antisense: 5′-GuAAGAcuuGAGAuGAuccT*T-3′where 2′-fluoro-modified nucleotides are in lower case andphosphorothioate linkages are represented by asterisks. Two dayspost-injection, a 100 ul blood sample was obtained from mice andcentrifuged at 13,000 rpm in serum separator tubes (Becton Dickinson).Serum levels of Factor VII were analyzed using a Biophen FVII assay kitas described previously (Aniara Corporation, Mason, Ohio) Semple, S. C.et al. Nature Biotechnology 1-7 (2010). Results shown in Table 2.

TABLE 2 Original Library FVII Activity Data 5 mg/kg 2 mg/kg 0.5 mg/kg0.1 mg/kg Rela- Rela- Rela- Rela- tive Stan- tive Stan- tive Stan- tiveStan- FVII dard FVII dard FVII dard FVII dard Lip- Ac- Devi- Ac- Devi-Ac- Devi- Ac- Devi- idoid tivity ation tivity ation tivity ation tivityation 64O14 0.92 0.05 68O10 0.91 0.20 68O11 1.03 0.11 77O13 0.85 0.1077O14 0.74 0.02 80O13 0.66 0.23 81O13 0.43 0.18 86O12 0.87 0.07 87O130.77 0.03 94O14 0.13 0.06 99O11 0.53 0.07 109O11 0.85 0.05 109O12 0.820.06 109O13 0.38 0.09 110O10 0.35 0.11 110O13 0.75 0.07 113O10 0.15 0.06113O11 0.57 0.20 113O12 0.01 0.00 0.09 0.10 0.49 0.08 0.59 0.08 113O130.00 0.00 0.00 0.00 0.04 0.11 0.48 0.12 113O14 0.75 0.14 120O11 0.930.04 120O12 0.88 0.15 120O13 0.92 0.08 120O14 0.79 0.05 122O10 0.13 0.06122O12 0.82 0.06 123O12 0.93 0.07 123O13 0.00 0.00 0.00 0.00 0.50 0.220.86 0.13 134O13 0.89 0.16 144O13 0.93 0.17 154O12 0.99 0.11 156O11 0.770.09 156O12 1.06 0.06 158O14 0.91 0.04 159O14 0.80 0.01 161O14 0.85 0.05164O14 0.90 0.08 166O10 0.99 0.03 166O14 0.83 0.08 191O11 0.61 0.19191O12 0.80 0.19 191O14 0.61 0.10 193O10 0.70 0.12 193O11 0.61 0.13193O12 0.70 0.09 195O12 0.43 0.07 195O13 0.56 0.04 196O13 0.80 0.01196O14 0.82 0.06 200O10 0.56 0.17 200O11 0.67 0.07 200O12 0.62 0.00200O13 0.40 0.10 200O14 0.91 0.20 205O12 0.78 0.10 205O14 0.83 0.09217O12 0.58 0.13 217O13 0.02 0.01 0.33 0.28 0.85 0.13 0.92 0.12 218O130.73 0.16 219O13 0.00 0.00 235O13 0.39 0.07 25O13 0.25 0.23 302O13 1.030.22 302O14 0.84 0.29 303O10 0.95 0.26 303O12 0.01 0.01 0.20 0.09 0.910.04 0.95 0.05 304O11 0.57 0.02 304O12 0.81 0.24 304O13 0.01 0.00 0.020.01 0.23 0.06 0.54 0.26 304O14 0.58 0.13 305O12 0.75 0.11 305O13 0.000.00 0.06 0.04 0.25 0.06 0.96 0.17 306O10 0.00 0.00 0.00 0.01 0.23 0.070.69 0.13 306O11 0.02 0.01 0.00 0.00 0.40 0.15 0.59 0.17 306O12 0.000.00 0.00 0.00 0.20 0.15 0.37 0.02 306O13 0.00 0.00 0.00 0.01 0.04 0.070.71 0.14 306O14 0.85 0.05 313O10 0.00 0.00 0.21 0.11 0.74 0.10 1.040.13 313O11 0.01 0.02 0.38 0.16 0.75 0.05 0.73 0.07 313O12 0.01 0.040.52 0.25 0.89 0.33 0.95 0.26 313O13 0.01 0.01 0.09 0.04 0.09 0.08 0.860.24 313O14 0.67 0.04 315O12 0.99 0.39 31O14 0.88 0.14 32O14 0.80 0.06347O10 0.92 0.08 347O11 0.85 0.16 347O12 0.15 0.08 347O13 0.67 0.2236O14 0.69 0.25 371O11 0.78 0.09 371O12 0.78 0.06 371O14 0.85 0.03Biodistribution and Immunostaining

Female C57BL/6 mice received tail vein injections of lipid nanoparticlescontaining siRNA that had been labeled with Cy5.5 on the 5′ end of thesense strand (provided by Alnylam Pharmaceuticals). Animals were dosedat 1 mg/kg of siRNA and volume of 0.01 ml/g. At one hour post-injection,mice were euthanized and organs were removed. Body-wide biodistributionwas assessed by imaging whole organs with an IVIS® Spectrum system(Caliper Life Sciences, Hopkinton, Mass.) at excitation and emissionwavelengths of 675 nm and 720 nm, respectively. Cell-specificdistribution within hepatocytes was assessed by embedding, sectioning,and staining the whole liver with antibodies. Imaging was conducted on aLSM 700 confocal microscope (Carl Zeiss, Inc., Peabody, Mass.). ForOdyssey and confocal imaging, organs were snap frozen on dry ice andembedded in optimal cutting temperature compound (OCT, LifeTechnologies, Grand Island, N.Y.). Cryostat sections were cut andcollected on superfrost plus treated slides. Prepared frozen sectionswhere kept at −20° C. until needed. Odyssey imaging was conducted on 20μm thick cryosections of tissue at a resolution of 21 μm (Lee, M. J.-E.et al., Rapid Pharmacokinetic and Biodistribution Studies UsingCholorotoxin-Conjugated Iron Oxide Nanoparticles: A NovelNon-Radioactive Method. PLoS ONE 5, e9536-e9536 (2010)).

For confocal imaging, liver tissue was cryosectioned (12 μm) and fixedusing 4% paraformaldehyde at room temperature for 30 min. All solutionswere prepared in PBS. Sections were washed 2× with PBS, permeabilizedfor 30 min with 0.1% Triton X100, and blocked for 1 hour with 5% normalgoat serum. Sections then incubated for 1 hour in an immunostainingcocktail solution consisting of DAPI (3 μM), Alexa Fluor 488 conjugatedanti-mouse F4/80 (1:200 dilution, BioLegend, San Diego, Calif.), AlexaFluor® 555 Phalloidin (1:200 dilution, Life Technologies), and 5% normalgoat serum. Slides were washed 3× with 0.1% Tween 20 and mounted usingProLong® Gold Antifade (Life Technologies). Sections were imaged usingan LSM 700 point scanning confocal microscope (Carl Zeiss, Inc, JenaGermany) equipped with a 40× oil immersion objective.

Blood Clearance

Blood clearance experiments were conducted by injecting LNPs containingCy5.5 -labeled siRNA at an siRNA dose of 0.5 mg/kg. Blood samples werecollected as a function of time via the retroorbital vein, with theexception of final time points, which were collected via cardiacpuncture. Serum, obtained by centrifugation, was diluted 1:30 in PBS andimaged and quantified using an Odyssey CLx imaging system (LI-CORBiosciences, Lincoln, Nebr.).

Histology

Organs were harvested from animals that had received various doses ofeither 304O₁₃ or C12-200 lipid nanoparticles (C12-200 is a controlnon-degradable lipidoid shown below). Organs were fixed overnight in 4%paraformaldehyde and transferred to 70% ethanol prior to paraffinembedding, sectioning, and H & E staining.

Serum Chemistry and Hematology Analysis

Post-sacrifice, cardiac sticks were immediately performed on animalsthat had been dosed with either 304O₁₃ or C12-200 lipid nanoparticles.Blood was centrifuged in serum separator tubes at 5,000 rpm for 10minutes, and serum was analyzed for various hematological parameters.Serum chemistry was evaluated on a Beckman Olympus AU400 Serum ChemistryAnalyzer. Cytokines were analyzed using Bio-Plex Pro Mouse Cytokine23-Plex Assay kits (Luminex Corporation, Austin, Tex.) on the Bio-Plex200 system, according to manufacturer instructions.

Cytokine Profiling

Cytokine analysis was done by injecting either 304O₁₃ or C12-200nanoparticles at an siRNA dose of 3 mg/kg. Four hours post-injection,blood was harvested via cardiac stick and serum was isolated. Cytokinelevels were quantified using an ELISA assay.

Nanoparticle Characterization

Lipid nanoparticles were diluted to an siRNA concentration of ˜5 ug/mlin 0.1×PBS, pH 7.3. siRNA entrapment efficiency was determined using theQuant-iT™ RiboGreen® RNA assay (Invitrogen). Particle sizes weremeasured with a ZETAPals analyzer (Brookhaven Instruments, Holtsville,N.Y.). Sizes reported are the average effective diameter of each LNP.Zeta potential measurements were acquired on a Zetasizer Nano ZS(Malvern, Westborough, Mass.), and reported values were the average of10-25 runs.

TABLE 3 Characterization Parameters for 304O₁₃ siRNA Entrapment DiameterZeta Potential (%) (nm) (mV) pKa 304O₁₃ 84.2 86.0 13.7 6.8 306O12 79.098.2 12.5 6.8 113O13 75.8 91.1 16.5 6.0Results and Discussion

Michael addition chemistry was employed to rapidly synthesize a libraryof 1400 lipid-like materials with the potential to serve as effective,biodegradable delivery vehicles (FIG. 1). 280 alkyl-amines (FIG. 2) werereacted combinatorially with 5 alkyl-acrylates to form lipidoidsconsisting of a polar, ionizable core surrounded by hydrophobic carbontails. Alkyl-amines, which were taken from commercially availablesupply, were chosen to maximize structural diversity and reactivitywithin a Michael addition scheme. We chose to work with alkyl-acrylatetails of intermediate length (10-14 carbon chain length), as previousstudies indicated that shorter tails often lack efficacy while longertails may cause insolubility during the nanoparticle formulation process(Akinc, A. et al. Nature Biotechnology 26, 561-569 (2008); Love, K. T.et al. Proc. Natl. Acad. Sci. USA 107, 1864-1869 (2010)).

The acrylate-based lipidoids provided herein also contain hydrolysableester moieties, functional groups which are commonly incorporated intodelivery vehicles to promote physiological degradation (Staubli, A.,Ron, E. & Langer, R. J. Am. Chem. Soc. 112, 4419-4424 (1990); vanDijkhuizen-Radersma, et al. Biomaterials 23, 4719-4729 (2002); Geng, Y.& Discher, D. E. J. Am. Chem. Soc. 127, 12780-12781 (2005)). Proton NMRanalysis indicated that a representative lipidoid, 304O₁₃, degraded tothe anticipated alkyl-alcohol product under hydrolytic conditions (FIGS.11A and 11B).

To determine the transfection ability of lipidoids, they were firstformulated into lipid nanoparticles (LNPs) containing siRNA, cholesteroland the helper lipids, DSPC and PEG(MW2000)-DMG. The delivery potentialof lipidoids was assessed by applying LNPs to HeLa cells that had beengenetically modified to stably express two reporter luciferase proteins:firefly and Renilla. Firefly luciferase served as the target gene whileRenilla luciferase served as a built-in control for toxicity andoff-targeting effects. Relative luciferase activity, which is the ratioof firefly to Renilla activity, is shown in FIG. 3 a after treatmentwith each LNP at an siRNA concentration of 40 nM. Of the 1400 members ofthe lipidoid library, ˜7% mediated target gene silencing of >50% (shownin red circles).

In order to extract structure-function information from the in vitrodata, we asked whether various structural properties were more or lesscommon within the group of efficacious lipidoids (red data points)compared to the bulk library. FIG. 3 b examines the importance of taillength on transfection. Because there were five tails used in thislibrary, each tail length made up 20% of the library. Of the LNPs thatwere effective in vitro, however, only 12% contained an O₁₀ tail.Occurrence rate (the y-axis value) was calculated as (the occurrencerate in the library)−(the occurrence rate in the group with >50%silencing). Therefore, the occurrence rate for O₁₀ is 12%−20%=−8%,indicating that it was significantly underrepresented among materialswith transfection potential. On the other hand, O₁₂ and O₁₃ tails wereoverrepresented in the efficacious group compared to the library atlarge, suggesting such tail lengths are associated with efficaciouslipidoids. FIG. 3 c suggests that lipidoids with the greatesttransfection potential were synthesized from alkyl-amines with three ormore substitution sites. The effect of various functional groups withinthe alkyl-amine is analyzed in FIG. 3 d. The presence of tertiary andsecondary amines, alcohols, and branched or linear chains conferredefficacy, while ethers and rings generally did not. Piperazine rings,however, were an exception, and generally produced efficaciousmaterials.

Previous studies have indicated that materials capable ofconferring >50% luciferase silencing activity in cell culture have thepotential to mediate siRNA delivery in vivo (Whitehead, K. A. et al. InVitro-In VivoTranslation of Lipid Nanoparticles for Hepatocellular siRNADelivery. ACS Nano 120706143602000 (2012).doi:10.1021/nn301922x).Selected lipidoids (those data points shown in red in FIG. 3 a) wereanalyzed for siRNA delivery to hepatocytes in a murine model of theblood coagulation Factor VII. The Factor VII model, which has beenwell-validated in the literature (Akinc, A. et al. Nature Biotechnology26, 561-569 (2008); John, M. et al. Nature 449, 745-747 (2007); Semple,S. C. et al. Nature Biotechnology 1-7 (2010)), allows silencing to beassessed from a few drops of blood using a commercially-available assay.In these experiments, LNPs containing anti-Factor VII siRNA wereinjected intravenously into mice, and Factor VII activity levels werequantified two days post-injection. Fifteen of the 108 lipidoidsanalyzed in vivo mediated complete knockdown of Factor VII proteinlevels at an siRNA dose of 5 mg/kg (FIG. 4 a). For these top LNPcandidates, control experiments conducted using non-targeting siRNA at 5mg/kg resulted in no FVII knockdown and suggested that reductions inprotein activity were not due to off-targeting or toxicity-mediated genedownregulation. Silencing for these top candidates was dose dependent(FIG. 8), with EC₅₀ values ranging from 0.05 to 2 mg/kg when LNPs wereformulated at a lipidoid:cholesterol:DSPC:PEG standard testing molarratio of 50:38.5:10:1.5.

While seeking an optimal molar ratio for the top LNPs (e.g. 306O₁₂,113O₁₃, and 304O₁₃), the PEG molar percentage was found to have aneffect on LNP efficacy. FIG. 4 c reveals that, for the lipidoid 304O₁₃,there is a range of PEG % between 0.5 and 1.0 where optimalhepatocellular delivery is achieved. The optimized 304O₁₃ formulation(PEG %=0.75) has an EC50 value, 0.01 mg/kg, that is a full order ofmagnitude lower than when using 1.5% PEG. Optimized 304O₁₃ behaved in adose dependent fashion (FIG. 4 d), and after a single injection at 0.1mg/kg, Factor VII levels returned to baseline within 18 days.

In addition to examining hepatocellular delivery, we also explored theability of biodegradable lipidoid materials to deliver siRNA toleukocyte populations in vivo. Immune cells are attractive targets forRNA interference therapy, as they have been implicated in variousaspects of disease initiation and progression, including inflammationand autoimmune responses (Geissmann, F. et al. Science 327, 656-661(2010); Grivennikov, et al. Cell 140, 883-899 (2010)). Although moderatelevels of gene silencing have been achieved recently in leukocytes(Leuschner, F. et al. Nature Biotechnology 29, 1005-1010 (2011);Novobrantseva, T. I. et al. Molecular Therapy—Nucleic Acids 1, e4(2012)), it will be important clinically that compounds can be degradedand eliminated from the body. In these experiments, LNPs were formulatedwith siRNA specific against CD45, which is a tyrosine phosphataseprotein found on the surface of all white blood cells. Three daysfollowing the intravenous delivery of LNPs in mice, immune cells wereharvested from the peritoneal cavity and spleen. Cells were stained withfluorescent antibodies, and CD45 protein silencing was quantified inspecific immune cell subsets via flow cytometry analysis. Results werenormalized to CD45 levels after delivery of the same LNP containing anon-targeting siRNA. Of the five lipidoid materials evaluated in thismodel, 304O₁₃ and 306O₁₃ mediated the most robust CD45 silencing inimmune cells isolated from both the peritoneal cavity and the spleen(FIGS. 4 e and f). CD11b+ and CD11c+ populations (monocyte/macrophagesand dendritic cells, respectively) were subject to high levels ofknockdown within the peritoneal cavity (up to 90%) and to a lesserdegree within the spleen (up to 40%). The lipidoids 306O₁₂, 306O₁₄, and315O₁₂ also offered modest CD45 silencing in several immune cellsubpopulations (FIG. 9).

Nanoparticle characterization parameters for three of the top LNPcandidates were similar (Table 1). Entrapment of siRNA refers to thepercentage of siRNA in solution that is incorporated into thenanoparticle during formulation, as measured by an RNA dye-binding assay(Nolan, T., Hands, R. E. & Bustin, S. A. Quantification of mRNA usingreal-time RT-PCR. Nat. Protoc 1, 1559-1582 (2006)). These results are inkeeping with a previous finding that efficacious lipidoid nanoparticlesoften have entrapment values of approximately 75%17. Zeta potentialmeasurements were conducted under neutral pH conditions. pKa values,which were obtained using a toluene nitrosulphonic acid (TNS) assay,evaluated the pKa of the nanoparticle surface (Heyes, J., Palmer, L.,Bremner, K. & MacLachlan, I. Cationic lipid saturation influencesintracellular delivery of encapsulated nucleic acids. J Control Release107, 276-287 (2005)). The pKa values of top LNP candidates corroboratethe results of another study in which surface pKa values in the 6-7range conveyed efficacy in vivo (Jayaraman, M. M. et al., Maximizing thePotency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo.Angew. Chem. Int. Ed. 51, 8529-8533 (2012)).

Several analyses were performed to assess the biodistribution of thelead compound, 304O₁₃, in mice. For these experiments, nanoparticleswere formulated with Cy5.5-labeled siRNA. Whole organ IVIS images (FIG.5 a) and Odyssey scans (FIG. 5 b) showed that naked siRNA accumulated inthe kidneys at 1 hour post-injection, suggesting rapid renal clearance.Quantification of IVIS signal indicated that 14%, 1%, and 71% of nakedsiRNA signal appeared in the liver, spleen, and kidneys, respectively.In contrast, at 1 hour post injection, 304O₁₃ localized primarily withinthe liver (42%) and spleen (24%), with only 18% distributing to thekidneys.

Given their effectiveness for silencing the hepatocellular target, FVII,we examined how 304O₁₃ nanoparticles were distributing within the liver.Confocal imaging was performed on liver tissues harvested one hourpost-injection and stained with nuclear, actin, and macrophage markers(FIG. 7 c). Images were taken near the central vein in liver lobules(black void near the center of images). Hepatocytes are outlined ingreen and macrophages, which appear sporadically, are colored magenta.Only 304O₁₃ was able to mediate siRNA accumulation throughout nearly allhepatocellular tissue (in red).

Serum clearance kinetics were assessed by measuring Cy5.5 signal in themouse bloodstream as a function of time (FIG. 5 d). It should be notedthat, while the first blood sample was drawn as quickly as possible (20seconds), maximum signal may have occurred even earlier. Half of thematerial initially detected at 20 seconds had distributed to tissues by6 minutes. At 90 minutes post-injection, only 4% of signal remained.

A preliminary safety assessment was conducted on the lead LNP, 304O₁₃,and it was compared to another previously-discovered LNP formulation,C12-200 (Love, K. T. et al. Lipid-like materials for low-dose, in vivogene silencing. PNAS 107, 1864-1869 (2010)). C12-200 is a 5-tailed,lipidoid that has the same EC₅₀ as 304O₁₃ (0.01 mg/kg). It was chosenfor comparison purposes because it does not contain any functionalgroups that are overtly sensitive to hydrolysis. We chose to examine theeffect of doses that were at least 100-fold higher than the EC₅₀. Serumcytokine levels for both materials were assessed in mice four hoursafter a 3 mg/kg IV bolus injection (total siRNA). IL-6, IP-10, KC, andMCP-1 were elevated in the C12-200 group compared to both PBS negativecontrol and 304O₁₃ groups under these conditions (FIG. 6). Clinicalchemistry parameters were evaluated for both materials 72 hours after asingle dose of 3 mg/kg and after four once weekly doses of 3 mg/kg each.There were no toxicologically significant increases in albumin, ALT,AST, ALP, total bilirubin, or GGT for either 304O₁₃ or C12-200 aftersingle or multiple doses (FIG. 12).

Histological analysis was performed through H&E staining on sectionsfrom the liver, spleen, kidneys and pancreas. In single-dose studies (0,1, 2, 3, 5, 7.5, 10 mg/kg), liver necrosis was observed in miceadministered ≧7.5 mg/kg of C12-200 and at 10 mg/kg of 304O₁₃. Pancreaticinflammation and islet cell enlargement were detected at C12-200 doses≧2 mg/kg. A small amount of apoptosis in splenic red pulp was observedat 10 mg/kg for 304O₁₃. Multi-dose studies were also conducted in whichmice received four injections of 0.3, 1, 2, 3, or 5 mg/kg, once per weekfor four weeks. Liver necrosis and inflammation were observed in miceadministered ≧1 mg/kg of C12-200. There was no sign of liver toxicity inany of the 304O₁₃ groups up to 5 mg/kg. Based on this limitedevaluation, the collective data suggest an improved toxicity profile for304O₁₃ compared to C12-200 in mice.

The data from the 108 materials tested in vivo at a total siRNA dose of5 mg/kg are shown in FIG. 7 a. Of the 108 materials tested in mice, 25of them contained an O₁₃ tail, 66 of them had three or more tails, and42 of them had been synthesized from an alkyl-amine that contained atleast one tertiary amine.

FIG. 7 b shows a second generation library of lipidoids from certainamines conjugated to an O₁₃ tail. When tested in vivo, 10 out of 12 ofthese materials mediated 100% Factor VII silencing at a dose of 5 mg/kg(FIG. 7 c). Knockdown was dose-dependent, with EC50 values varying from0.05-1 mg/kg (FIG. 7 d). Formulation optimization of the best secondgeneration material, 503O₁₃, markedly decreased the EC₅₀ value to 0.01mg/kg (FIG. 7 e). Several second generation materials also facilitatedsignificant CD45 knockdown in monocyte, macrophage, dendritic cell, andB cell populations (FIG. 13).

Since the ability of materials to take on a positive charge withdecreasing pH has been shown to confer transfection efficacy (Zhang, J.J., Fan, H. H., Levorse, D. A. D. & Crocker, L. S. L. Ionizationbehavior of amino lipids for siRNA delivery: determination of ionizationconstants, SAR, and the impact of lipid pKa on cationiclipid-biomembrane interactions. Langmuir 27, 1907-1914 (2011)), thesurface pKa values of 59 distinct lipidoid nanoparticles were measured.The data in FIG. 10 indicate that pKa values play a decisive role inthis LNP delivery system, with a critical pKa value of approximately5.5. Materials demonstrating considerable in vivo efficacy (red datapoints) had surface pKa values of approximately 5.5 or higher. Forvalues less than approximately 5.5, average efficacy decreasedmonotonically with pKa. Therefore, surface pKa can be used as anindicator of in vivo potency, improving our predictive capability forthis data set.

Other Embodiments

All patents, patent applications, and literature references cited hereinare incorporated herein by reference.

Having now described some illustrative embodiments of the invention, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the invention. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction. Use of terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

What is claimed is:
 1. A compound of Formula (I):

or a salt thereof, wherein each L is, independently, branched orunbranched C₁₋₆ alkylene, wherein L is optionally substituted with oneor more fluorine radicals; each R^(A) is, independently, branched orunbranched C₁₋₆ alkyl, C₃₋₇ cycloalkyl, or branched or unbranched C₄₋₁₂cycloalkylalkyl, wherein R^(A) is optionally substituted with one ormore fluorine radicals; each R is, independently, hydrogen, or—CH₂CH₂C(═O)OR^(B), provided that at least three R groups are—CH₂CH₂C(═O)OR^(B); each R^(B) is, independently, C₁₀₋₁₄ alkyl, whereinR^(B) is optionally substituted with one or more fluorine radicals; andq is 1, 2, or 3; provided that the compound is not


2. The compound of claim 1, wherein the compound is of the Formula(I-a):

or a salt thereof, wherein each n is, independently, 0, 1, or 2; and mis 0, 1, or
 2. 3. The compound of claim 1, wherein L is unbranched C₁₋₆alkylene.
 4. The compound of claim 1, wherein the compound is of theformula

or a salt thereof.
 5. The compound of claim 1, wherein the compound isof the formula:

or a salt thereof.
 6. The compound of claim 1, wherein all R groups are—CH₂CH₂C(═O)OR^(B).
 7. The compound of claim 1, wherein all R^(B) groupsare the same.
 8. The compound of claim 1, wherein R^(B) is C₁₀ alkyl,C₁₁ alkyl, C₁₂ alkyl, C₁₃ alkyl, or C₁₄ alkyl.
 9. A compound selectedfrom the group consisting of:

and salts thereof.
 10. A nanoparticle comprising a compound of claim 1and one or more agents to be delivered.
 11. The nanoparticle of claim10, wherein the one or more agents is a polynucleotide, drug, protein,peptide, a small molecule, or gas.
 12. A composition comprising one ormore compounds of claim 1, and an excipient.
 13. The composition ofclaim 12, wherein the composition further comprises an agent selectedfrom the group consisting of an organic molecule, inorganic molecule,nucleic acid, protein, peptide, polynucleotide, targeting agent, anisotopically labeled chemical compound, vaccine, and an immunologicalagent.
 14. A method of administering an agent, the method comprising:administering to a subject in need thereof a therapeutically effectiveamount of a composition comprising a compound of claim 1 and an agent tobe delivered.
 15. The compound of claim 1, wherein L is ethylene. 16.The compound of claim 1, wherein L is propylene.
 17. The compound ofclaim 1, wherein q is
 1. 18. The compound of claim 1, wherein q is 2.19. The compound of claim 1, wherein q is
 3. 20. The compound of claim1, wherein R^(A) is branched or unbranched C₁₋₆ alkyl.
 21. The compoundof claim 1, wherein R^(A) is methyl, ethyl, or propyl.
 22. The compoundof claim 1, wherein R^(A) is C₃₋₇ cycloalkyl.
 23. The nanoparticle ofclaim 11, wherein the one or more agents is a polynucleotide, and thepolynucleotide is RNA.
 24. The composition of claim 13, wherein theagent is a polynucleotide, and the polynucleotide is DNA.
 25. Thecomposition of claim 13, wherein the agent is a polynucleotide, and thepolynucleotide is RNA.
 26. The composition of claim 25, wherein the RNAis mRNA, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.
 27. Thecomposition of claim 13, wherein the agent is a polynucleotide, and thepolynucleotide encodes a protein or peptide.